2-D Electrophoresis - University of Alberta

2-D Electrophoresis - University of Alberta
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2-D Electrophoresis – Principles and Methods
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2-D Electrophoresis
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2-D Electrophoresis
Principles and Methods
80-6429-60 AD 1
Preface
Despite alternative technologies that have emerged, 2-dimensional (2-D) electrophoresis is
currently the only technique that can be routinely applied for parallel quantitative expression
profiling of large sets of complex protein mixtures. Furthermore, it delivers a map of intact
proteins that reflects changes in protein expression level, isoforms, or post-translational
modifications. Last but not least, today’s 2-D electrophoresis technology with immobilized
pH gradients (IPGs) has overcome the former limitations of carrier-ampholyte-based 2-D
electrophoresis with respect to reproducibility, handling, resolution, and separation of very
acidic and/or basic proteins (NEPHGE). The development of IPGs up to pH 12 together with an
optimized protocol has enabled the analysis of very alkaline proteins and the construction
of the corresponding databases. Narrow-overlapping IPGs provide increased resolution
(∆pI = 0.001) and, in combination with prefractionation methods, the potential for the
detection of low-abundance proteins.
The technique of 2-D electrophoresis with IPG strips has been constantly refined. It is now
readily available to many laboratories and is more or less routine. Moreover, Difference
Gel Electrophoresis (DIGE) has proved to be a most powerful and exciting technique for the
reliable detection and quantitation of differentially expressed proteins. However, there are
still challenges with respect to proteomic samples that span an immense dynamic range
of relative abundance and a variety of physicochemical properties including solubility,
hydrophobicity/hydrophilicity, size, and/or charge. Consequently, sample preparation and
prefractionation are actually in the focus of interest, combined with new instrumentation
for multiple runs and high-throughput analysis. Is there perfection in view? There are still
some challenges in the state-of-the-art technology of 2-D electrophoresis but less than often
expected and repeatedly described.
It is my pleasure to introduce the third edition of a most successful manual on 2-D electrophoresis.
It clearly describes the actual techniques for 2-D electrophoresis with IPG strips, which should be
stringently controlled, and provides detailed protocols for newcomers as well as for experienced
users. New techniques such as 2-D DIGE and different sample preparation methods are included.
Finally, there is a most valuable comprehensive pictorial troubleshooting guide — just in case
(Murphy’s Law!) something went wrong.
Angelika Görg
Technical University of Munich, Germany
September 2004
2 80-6429-60 AD
Contents
Preface..............................................................................................................................................................2
Introduction....................................................................................................................................................7
Introduction to this handbook.......................................................................................................................................7
Introduction to 2-D electrophoresis............................................................................................................................7
Symbols used in this handbook..........................................................................................................................8
A. First- and second-dimension electrophoresis with optimized systems...................................9
B. First- and second-dimension electrophoresis with a flatbed system..................................... 11
Equipment choices........................................................................................................................................................... 12
Selecting an IEF system....................................................................................................................................... 12
Selecting a second-dimension system........................................................................................................ 13
Vertical systems....................................................................................................................................................... 14
Multiphor II Electrophoresis System.............................................................................................................. 14
Good laboratory practice.............................................................................................................................................. 15
1. Sample preparation.................................................................................................................................17
1.0 General strategy...................................................................................................................................................... 17
1.0.1 Cell disruption, protection from proteolysis, fractionation.................................................. 17
1.0.2 Precipitation and removal of interfering substances............................................................. 17
1.0.3 Additional aspects of sample preparation.................................................................................. 18
1.0.4 General sample preparation guidelines........................................................................................ 19
1.1 Methods of cell disruption.................................................................................................................................. 20
1.1.1 Gentle lysis methods............................................................................................................................... 20
1.1.2 More vigorous lysis methods.............................................................................................................. 21
1.1.3 Processing small tissue or cell samples using Sample Grinding Kit............................... 21
1.1.4 Preparing samples from “difficult” protein sources................................................................ 22
1.2 Protecting against proteolysis ........................................................................................................................ 23
1.2.1 Protease inhibition using Protease Inhibitor Mix...................................................................... 24
1.3 Fractionation of protein lysates....................................................................................................................... 24
1.4 Precipitation procedures..................................................................................................................................... 25
1.4.1 Cleaning up samples using 2-D Clean-Up Kit............................................................................. 26
1.4.2 Resuspension of pellet........................................................................................................................... 29
1.5 Other methods for removing contaminants............................................................................................. 30
1.5.1 Desalting samples using Mini Dialysis Kit.................................................................................... 32
1.5.2 Removing undesirable nucleic acids from samples using Nuclease Mix..................... 34
1.5.3 Simultaneous DNA, RNA and protein isolation from undivided scarce samples...... 34
1.5.4 Using Albumin and IgG Removal Kit to improve
2-D electrophoresis of human serum............................................................................................ 34
1.6 Composition of sample preparation solution........................................................................................... 37
1.6.1 Components of sample preparation solutions.......................................................................... 37
1.6.2 Examples of sample preparation solutions................................................................................. 38
1.7 Quantitating protein samples........................................................................................................................... 38
1.7.1 Protein determination using 2-D Quant Kit................................................................................. 39
1.8 Sample loads............................................................................................................................................................. 40
2. First-dimension isoelectric focusing (IEF)...........................................................................................41
2.0 Overview...................................................................................................................................................................... 41
2.1 Background to isoelectric focusing............................................................................................................... 41
2.2 Immobiline DryStrip gels .................................................................................................................................... 43
2.2.1 Choosing strip length............................................................................................................................. 44
80-6429-60 AD 3
2.2.2 Choosing the pH gradient.................................................................................................................... 45
2.2.3 Choosing an IPG Buffer.......................................................................................................................... 46
2.2.4 Estimating the pI of proteins............................................................................................................... 46
2.3 IEF using Ettan IPGphor 3 Isoelectric Focusing System and accessories................................... 47
2.3.2 Ettan IPGphor 3 Manifold...................................................................................................................... 48
2.3.3 IPGbox............................................................................................................................................................ 51
2.3.4 Ettan IPGphor 3 Strip Holders ........................................................................................................... 51
2.3.5 General cautions ..................................................................................................................................... 51
2.4 Selecting sample application method.......................................................................................................... 52
2.4.1 Rehydration loading .............................................................................................................................. 52
2.4.2 Use of Manifold.......................................................................................................................................... 52
2.4.3 Paper-bridge loading ............................................................................................................................ 52
2.5 Recommended sample loads........................................................................................................................... 53
2.6 Immobiline DryStrip gel rehydration solutions......................................................................................... 53
2.6.1 Components of rehydration solution............................................................................................. 54
2.6.2 Using DeStreak Rehydration Solution............................................................................................ 55
2.6.3 Preparation of other rehydration solutions................................................................................. 57
2.7 Immobiline DryStrip Gel rehydration using accessories..................................................................... 57
2.8 Isoelectric focusing guidelines—Ettan IPGphor 3 System.................................................................. 63
2.8.1 Protocol examples—Ettan IPGphor 3 Isoelectric Focusing System................................. 63
2.8.2 Running an Ettan IPGphor 3 protocol............................................................................................. 63
2.8.3 Preservation of focused Immobiline DryStrip gels.................................................................. 67
2.9 Troubleshooting....................................................................................................................................................... 68
3. Second-dimension SDS-PAGE using vertical electrophoresis systems........................................71
3.0 Overview...................................................................................................................................................................... 71
3.1 Equilibrating Immobiline DryStrip gels......................................................................................................... 71
3.1.1 Equilibration solution components................................................................................................. 71
3.1.2 Equilibrating Immobiline DryStrip gels.......................................................................................... 72
3.2 Background to SDS-PAGE.................................................................................................................................... 72
3.3 Electrophoresis using Ettan DALT Large Vertical electrophoresis systems............................... 73
3.3.1 Preparing Ettan DALT system for electrophoresis using precast gels............................74
3.3.2 Inserting DALT Gel 12.5 into DALT Precast Gel Cassette....................................................... 76
3.3.3 Equilibrating Immobiline DryStrip gels ......................................................................................... 77
3.3.4 Applying equilibrated Immobiline DryStrip gels to SDS gels............................................... 77
3.3.5 Inserting gels into Ettan DALT electrophoresis units.............................................................. 78
3.3.6 Electrophoresis conditions with precast gels for both
Ettan DALTsix and Ettan DALTtwelve............................................................................................... 80
3.3.7 Preparing lab-cast gels......................................................................................................................... 80
3.3.8 Preparing Ettan DALT electrophoresis units for electrophoresis
sing lab-cast gels..................................................................................................................................... 83
3.3.9 Equilibrating Immobiline DryStrip gels with lab-cast gels................................................... 83
3.3.10 Applying Immobiline DryStrip gels to lab-cast gels................................................................ 83
3.3.11 Inserting lab-cast gels into Ettan DALT electrophoresis units........................................... 83
3.3.12 Electrophoresis conditions with lab-cast gels........................................................................... 83
3.3.13 Troubleshooting........................................................................................................................................ 83
3.4 Electrophoresis using other vertical electrophoresis systems........................................................ 84
3.4.1 Preparing caster and gel sandwich for miniVE, SE 260,
and SE 600 Ruby electrophoresis systems.................................................................................. 84
3.4.2 Preparing lab-cast gels for miniVE, SE 260,
and SE 600 Ruby electrophoresis systems.................................................................................. 84
4 80-6429-60 AD
3.4.3 Preparing miniVE, SE 260, and SE 600 Ruby systems for electrophoresis................... 86
3.4.4 Equilibrating Immobiline DryStrip gels.......................................................................................... 86
3.4.5 Applying Immobiline DryStrip gels................................................................................................... 87
3.4.6 Inserting gels into miniVE, SE 260, and SE 600 Ruby systems............................................ 87
3.4.7 Electrophoresis conditions.................................................................................................................. 87
3.5 Troubleshooting....................................................................................................................................................... 88
4. Use of the flatbed Multiphor II Electrophoresis System for first and second dimensions........91
4.0 Overview...................................................................................................................................................................... 91
4.1 First-dimension IEF using Multiphor II Electrophoresis System and
Immobiline DryStrip Kit........................................................................................................................................ 91
4.1.1 Immobiline DryStrip gel rehydration—IPGbox........................................................................... 92
4.1.2 Preparing for IEF........................................................................................................................................ 93
4.1.3 Sample application by cup loading................................................................................................. 94
4.1.4 Paper-bridge loading.............................................................................................................................. 95
4.1.5 IEF guidelines for Multiphor II Electrophoresis System.......................................................... 96
4.1.6 Protocol examples................................................................................................................................... 96
4.1.7 Running a Multiphor II protocol......................................................................................................... 97
4.1.8 Preservation of focused Immobiline DryStrip gels.................................................................. 99
4.1.9 Troubleshooting......................................................................................................................................100
4.2 Second-Dimension SDS-PAGE using Multiphor II Electrophoresis System...............................101
4.2.1 ExcelGel preparation............................................................................................................................101
4.2.2 Applying equilibrated Immobiline DryStrip gels......................................................................102
4.2.3 Electrophoresis conditions................................................................................................................103
4.2.4 Troubleshooting......................................................................................................................................104
5. Visualizing and evaluating results.................................................................................................... 105
5.0 Visualizing results—labeling and staining................................................................................................105
5.0.1 Automating processing and preserving the gel.....................................................................106
5.1 Blotting.......................................................................................................................................................................107
5.2 Evaluating results.................................................................................................................................................107
5.3 Standardizing results..........................................................................................................................................108
5.4 Further analysis of protein spots..................................................................................................................108
5.4.1 Picking protein spots.............................................................................................................................108
5.4.2 Digesting proteins and spotting onto MALDI-ToF MS slides..............................................108
5.4.3 MALDI-ToF mass spectrometry.......................................................................................................108
6. 2-D Fluorescence Difference Gel Electrophoresis (2-D DIGE)...................................................... 111
6.0 Overview....................................................................................................................................................................111
6.1 CyDye DIGE Fluor dyes........................................................................................................................................113
6.1.1 CyDye DIGE Fluor minimal dyes......................................................................................................113
6.1.2 Minimal labeling of protein with CyDye DIGE Fluor minimal dyes..................................114
6.2 CyDye DIGE Fluor labeling kits with saturation dyes for
labeling scarce samples and preparative gels......................................................................................114
6.3 Ettan DIGE system workflow...........................................................................................................................116
6.3.1 Experimental design for Ettan DIGE system applications..................................................117
6.3.2 Sample preparation for Ettan DIGE system applications...................................................119
6.3.3 Sample labeling with minimal dyes for Ettan DIGE system applications...................120
6.3.4 Two-dimensional separation of protein samples...................................................................121
6.3.5 Summary of key differences between minimal labeling and saturation labeling .122
6.3.6 Imaging.......................................................................................................................................................122
6.3.7 Image analysis with DeCyder 2-D Differential Analysis Software.................................123
6.3.8 Further analysis of protein spots....................................................................................................123
6.4 Troubleshooting 2-D DIGE.................................................................................................................................124
80-6429-60 AD 5
7. Troubleshooting..................................................................................................................................... 125
.......................................................................................................................................... 129
Appendix I
Solutions..............................................................................................................................................................................129
A. Sample preparation solution (with urea) for 2-D electrophoresis...........................................129
B. Sample preparation solution (with urea and thiourea) for 2-D electrophoresis..............129
C. Urea rehydration stock solution...............................................................................................................130
D. Thiourea rehydration stock solution......................................................................................................130
E. SDS equilibration buffer solution..............................................................................................................130
F. 10× Laemmli SDS electrophoresis buffer..............................................................................................131
G. 30% T, 2.6% C monomer stock solution...............................................................................................131
H. 4× resolving gel buffer solution................................................................................................................131
I. Bromophenol blue stock solution..............................................................................................................131
J. 10% SDS solution..............................................................................................................................................131
K. 10% ammonium persulfate solution......................................................................................................132
L. Gel storage solution........................................................................................................................................132
M. 1× Laemmli SDS electrophoresis buffer..............................................................................................132
N. Agarose sealing solution.............................................................................................................................132
Appendix II.................................................................................................................................................. 133
Optimized silver staining of large-format DALT gels and
DALT 12.5 precast gels using PlusOne Silver Staining Kit, Protein...........................................................133
Appendix III................................................................................................................................................. 135
Colloidal Coomassie staining procedure.............................................................................................................135
5% Coomassie Blue G-250 stock...................................................................................................................135
Colloidal Coomassie Blue G-250 dye stock solution............................................................................135
Colloidal Coomassie Blue G-250 working solution...............................................................................135
Appendix IV................................................................................................................................................. 137
Appendix V.................................................................................................................................................. 141
Treating glass plates with Bind-Silane..................................................................................................................141
Appendix VI................................................................................................................................................. 143
Using Ready-Sol...............................................................................................................................................................143
References.................................................................................................................................................. 145
Additional reading and reference material......................................................................................... 149
Ordering information............................................................................................................................... 151
Recommended additional consumables.............................................................................................. 159
6 80-6429-60 AD
Introduction
Introduction to this handbook
This handbook is intended to provide guidelines for performing high-resolution two-dimensional (2-D) electrophoresis.
It is divided into seven chapters:
Chapter 1 provides guidelines for sample preparation and protein quantitation.
Chapter 2 details procedures for performing the first dimension of 2-D electrophoresis, highlighting use of
Ettan™ IPGphor™ 3 Isoelectric Focusing System.
Chapter 3 contains general directions for subsequent second-dimension electrophoresis of immobilized pH gradient
(IPG) strips using various vertical gel electrophoresis systems.
Chapter 4 describes use of the flatbed Multiphor™ II Electrophoresis System for both first- and second-dimension
electrophoresis.
Chapter 5 discusses visualization and analysis of 2-D electrophoresis results.
Chapter 6 describes the advantages and use of the technique of 2-D Fluorescence Difference Gel Electrophoresis (2-D DIGE).
Chapter 7 describes common problems in 2-D gel electrophoresis and their remedies. Technique-specific
troubleshooting guides are included in the relevant chapters.
The protocols described in this handbook are performed using products from Amersham Biosciences, now a part of
GE Healthcare (referred to hereafter as GE Healthcare). Equipment choices are illustrated in Table 1. Product ordering
information is given on page 155.
Depending on the sample type and the nature of the investigation, the procedures may need to be adjusted or optimized.
Introduction to 2-D electrophoresis
2-D electrophoresis is a powerful and widely used method for the analysis of complex protein mixtures extracted from
cells, tissues, or other biological samples. This technique separates proteins according to two independent properties
in two discrete steps.
The first-dimension step, isoelectric focusing (IEF), separates proteins according to their isoelectric points (pI); the
second-dimension step, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), separates proteins
according to their molecular weights (Mr, relative molecular mass). Each spot on the resulting two-dimensional
gel potentially corresponds to a single protein species in the sample. Thousands of different proteins can thus be
separated, and information such as the protein pI, the apparent molecular weight, and the amount of each protein
can be obtained.
Two-dimensional electrophoresis was first introduced by O’Farrell (1) in 1975. In the original technique, the firstdimension separation was performed in carrier-ampholyte-containing polyacrylamide gels cast in narrow tubes. See
section 2.1 on page 43 for more details.
The power of 2-D electrophoresis as a biochemical separation technique has been recognized virtually since its
introduction. Its application, however, has become significant as a result of a number of developments:
• The introduction of immobilized pH gradients and Immobiline™ reagents (2) brought superior resolution and
reproducibility to first-dimension IEF. Based on this concept, Görg and colleagues (3,4) developed the currently
employed 2-D technique, where carrier-ampholyte-generated pH gradients have been replaced with immobilized pH
gradients, and tube gels replaced with gels supported by a plastic backing. A more detailed discussion of the merits
of this technique is presented in section 2.2 on page 45.
• 2-D DIGE, first described in 1997 by Ünlü et al. (5), offers a method for controlling system variations, allowing
biological variations and changes in protein expression to be identified with statistical confidence.
80-6429-60 AD 7
• Automation of steps after 2-D electrophoresis, such as gel image analysis, spot picking, spot digestion, and target
preparation for mass spectrometry, have allowed a significant increase in the throughput of protein analysis and
identification.
• New mass spectrometry techniques have been developed that allow rapid identification and characterization
of very small quantities of peptides and proteins.
• More powerful, less expensive computers and software are now available, rendering thorough computerized
evaluations of highly complex 2-D patterns to become economically feasible.
• Data about entire genomes of a number of organisms are now available, allowing rapid identification of the gene
encoding a protein separated by 2-D electrophoresis.
• Protein sequences are being added on a daily basis to databases available on the public domain. Organizations
such as the Human Proteome Organization (HUPO) are attempting to coordinate proteome analysis between many
countries toward a common goal.
• The World Wide Web provides simple, direct access to spot-pattern databases for the comparison of
electrophoresis results and genome sequence databases for assignment of sequence information.
A large and growing application of 2-D electrophoresis is within the field of proteomics (6,7). The analysis involves
the systematic separation, identification, and quantitation of many proteins simultaneously from a single sample.
Two-dimensional electrophoresis is used in this field due to its unparalleled ability to separate thousands of proteins
simultaneously. The technique is also unique in its ability to detect post- and co-translational modifications, which
cannot be predicted from the genome sequence. Applications of 2-D electrophoresis include proteome analysis, cell
differentiation, detection of disease markers, therapy monitoring, drug discovery, cancer research, purity checks, and
microscale protein purification. This handbook describes methods for 2-D electrophoresis using precast IPG strips
(Immobiline DryStrip gels) available from GE Healthcare.
Symbols used in this handbook
This symbol indicates general advice that can improve procedures or provide recommendations for action under
specific situations.
This symbol denotes advice that should be regarded as mandatory and gives a warning when special care
should be taken.
This symbol highlights troubleshooting advice to help analyze and resolve difficulties that may occur.
chemicals, buffers and equipment
experimental protocol
8 80-6429-60 AD
Table 1. Equipment choices for 2-D electrophoresis.
A. First- and second-dimension electrophoresis with optimized systems
First-dimension IEF using Ettan IPGphor 3 Isoelectric Focusing System
Gel sizes are given as gel width × separation length.
Ettan IPGphor 3 Isoelectric Focusing System with Ettan IPGphor Manifold or Standard Strip Holder
Note: The original IPGphor is fully compatible with the Manifold and with the protocols described throughout this handbook.
Choice Factors:
• Unique design of Ettan IPGphor 3 Manifold allows IEF
of up to 12 IPG strips, from 7 to 24 cm in length, with
subsequent equilibration of the strips.
• Protein-focusing patterns can be improved using
cup-based sample application, particularly in basic
IPG strips.
• Manifold tray base is made of a thermally conductive
aluminum oxide ceramic that rapidly dissipates heat
to avoid “hot spots.”
• Voltage, current, temperature, and time controls are
programmable.
Fig 1. Ettan IPGphor 3 Isoelectric Focusing System.
• Integral control software—with an external personal
computer (Windows™) connected via a serial port—
can be used to control up to four Ettan IPGphor 3
units simultaneously, each running a different set of
run parameters.
• Up to 10 protocols (nine steps each) can be saved,
retrieved, and easily edited on the instrument.
Second-dimension SDS-PAGE using various vertical electrophoresis systems
Ettan DALTsix Large Vertical System, up to six 26 × 20 cm gels
Choice Factors:
• Four-hour to overnight electrophoresis.
• Modular system.
• Precast gels with stable buffer system available, cast
on film support: Ettan DALT Gel 12.5 (25.5 × 19.6 cm,
1 mm thickness).
• Large-format gels for highest resolution and
maximum protein load.
• Medium throughput (up to six gels simultaneously).
Fig 2. Ettan DALTsix Large Vertical System.
• Best for 18- and 24-cm IPG strips.
• Buffer volume approximately 5 l for six gels.
80-6429-60 AD 9
Ettan DALTtwelve Large Vertical System, up to twelve 26 × 20 cm gels
Choice Factors:
• Four-hour to overnight electrophoresis.
• Integrated system with very efficient Peltier
temperature control.
• Precast gels with stable buffer system available, cast
on film support: Ettan DALT Gel 12.5 (25.5 × 19.6 cm,
1 mm thickness),
• Large-format gels for highest resolution and
maximum protein load.
Fig 3. Ettan DALTtwelve Large Vertical System.
• High throughput (up to 12 gels simultaneously).
• Best for 18- and 24-cm IPG strips.
• Buffer volume approximately 10 l for 12 gels.
miniVE and SE 260 (Mini-Vertical), one or two 8 × 7 or 9.5-cm gels
Choice Factors:
• Rapid: 1–2 h electrophoresis.
• Best for 7-cm IPG strips.
• Ideal when quick profiling is required or when the
protein pattern is relatively simple.
Fig 4. miniVE Vertical Eelctrophoresis System.
Fig 5. SE 260.
10 80-6429-60 AD
SE 600 Ruby (standard vertical), one to four 14 × 16 cm gels
Choice Factors:
• Electrophoresis in 2–5 h.
• Intermediate separation (16-cm gel length).
• Intermediate throughput (up to four gels
simultaneously using divider plates).
• Best for 11- or 13-cm IPG strips.
• Optional short plates for higher throughput of
7-cm IPG strips (up to eight strips per run using divider
plates).
Fig 6. SE 600 Ruby.
B. First- and second-dimension electrophoresis with a flatbed system
First-dimension IEF using Multiphor II Electrophoresis System with Immobiline DryStrip Kit
Rehydration in IPGbox
Choice Factors:
• Can be used for both first- and second-dimension
separations, as well as for many other electrophoresis
techniques.
• Versatile system for IEF with IPG strips from 7 to 24 cm.
• Note: EPS 3501 XL Power Supply and MultiTemp™ III
Thermostatic Circulator are required to supply power
and cool the system, respectively.
Fig 7. Multiphor II Electrophoresis System with Immobiline DryStrip Kit.
Second-dimension SDS-PAGE using Multiphor II Electrophoresis System, one 24.5 × 11/18 cm gel
Choice Factors:
• Precast gels available: ExcelGel™ SDS Homogeneous 12.5 (24.5 × 11 cm) and
ExcelGel Gradient XL 12–14 (24.5 × 18 cm).
• Relatively rapid: 4 h or less for electrophoresis.
• High resolution.
All available IPG strip lengths can be used.
80-6429-60 AD 11
The experimental sequence for 2-D electrophoresis is:
1. Sample preparation
Proper sample preparation is absolutely essential for good 2-D results.
2. Immobiline DryStrip gel rehydration
Immobiline DryStrip gels must be rehydrated with the appropriate additives prior to IEF.
3. IEF
First-dimension IEF is performed on a flatbed system at very high voltages with active temperature control.
4. Immobiline DryStrip gel equilibration
Strip equilibration in SDS-containing buffer prepares the sample for the second-dimension separation.
5. SDS-PAGE
The strip is placed on the second-dimension gel for SDS-PAGE.
6.
Visualization
Protein spots are stained to visualize them in the second-dimension gel matrix. Alternatively, if the proteins were
prelabeled, the spots can be visualized by autoradiography, by illumination of the gel with UV light, or by using a
fluorescence imager to detect the proteins.
7. Analysis
Analysis of the resultant two-dimensional array of spots.
Equipment choices
There are different options for methods and equipment for IEF and SDS-PAGE. Table 1 lists the instruments available
from GE Healthcare. For detailed information on the operation of any of the instruments described, refer to the
respective instrument user manual. For other details about the instruments and related products, refer to the
GE Healthcare BioDirectory or visit www.gehealthcare.com.
Selecting an IEF system
GE Healthcare offers two systems for first-dimension separation: Ettan IPGphor 3 Isoelectric Focusing System and
Multiphor II Electrophoresis System. Both are available with accessories for improving IEF performance.
The upgraded, easy-to-use Ettan IPGphor 3 Isoelectric Focusing System (Fig 1) simplifies the first-dimension
separation with a system dedicated to IEF on Immobiline DryStrip gels. Ettan IPGphor 3 consistently delivers speed
and reproducibility, and can handle high protein loads. The system incorporates a safe, high-voltage (up to 10 000
V, depending on the DryStrip being used) power supply and Peltier solid-state temperature control (15–30 °C).
Programmable parameters include rehydration temperature and duration, IEF temperature and maximum current,
and the duration and voltage pattern of multiple steps for each separation.
In addition to the IEF unit, key accessories include Ettan IPGphor Manifold, Strip Holders, and IPGbox. Integral Ettan
IPGphor 3 Control Software provides greater control in IEF runs; it can be used to control up to four Ettan IPGphor 3
units simultaneously, each running a different set of parameters. These accessories are discussed in detail in section 2.3.
For gradients at the upper and lower ends of the pH scale, as well as for very high protein loads on narrow-pHrange gradient strips, Ettan IPGphor 3 Manifold is employed for IEF using 7-, 11-, 13-, 18-, and 24-cm Immobiline
DryStrip gels. Samples can be loaded onto IPG strips using sample cups, Ettan IPGphor 3 IPGbox, or paper
bridges. Sections 2.3–2.5 discuss these options.
The versatile Multiphor II Electrophoresis System (Fig 7) can be used to perform several different electrophoresis
techniques. An advantage of the Multiphor II Electrophoresis System for 2-D electrophoresis is the fact that it can
be used for both first-dimension IEF and second-dimension SDS-PAGE. Strip rehydration with or without samples
is performed in the Immobiline DryStrip IPGbox. After rehydration, the Immobiline DryStrip gels are transferred to the
electrophoresis unit for first-dimension IEF.
12 80-6429-60 AD
The system is composed of the Multiphor II Electrophoresis System and Immobiline DryStrip Kit, which
also allows cup and paper-bridge loading of the sample onto rehydrated Immobiline DryStrip gels (using
Immobiline DryStrip IPGbox). This system accommodates up to 12 rehydrated Immobiline DryStrip gels of
the same length for any one IEF protocol. Power is supplied by the separate EPS 3501 XL power supply, and
temperature control by the separate MultiTemp III Thermostatic Circulator.
Table 2 shows the key operating differences between the Ettan IPGphor 3 Isoelectric Focusing System and Multiphor II
Electrophoresis System for first-dimension IEF.
Table 2. IEF system selection.
Ettan IPGphor 3
Maximum voltage
10 000 V
3500 V†
Multiphor II
Additional equipment required
Time required for IEF*
Manifold plus Immobiline DryStrip IPGbox
2–36 h
Immobiline DryStrip IPGbox
Immobiline DryStrip Kit,
EPS 3501 XL Power Supply,
MultiTemp III Thermostatic Circulator
2–72 h
* Optimal focusing time varies widely depending on the Immobiline DryStrip gel length and pH range, and the nature of the sample.
Similar separations can generally be performed at least two-fold faster with the Ettan IPGphor 3 Isoelectric Focusing System than
with the Multiphor II Electrophoresis System.
†
Higher voltages are not recommended for safety reasons.
Guidelines for the selection of sample application methods for Ettan IPGphor 3 Isoelectric Focusing System and
Multiphor II Electrophoresis System and can be found in sections 2.4 and 4.1.3–4.1.4, respectively.
Selecting a second-dimension system
The second-dimension separation may be performed in a vertical or flatbed system. Table 3 lists the appropriate
second-dimension system for a given gel size and Immobiline DryStrip gel length. Further considerations are
discussed below. For a more complete discussion of the relative merits of vertical compared with flatbed seconddimension systems, see reference 8.
Table 3. Selection of second-dimension electrophoresis system with suggested Immobiline DryStrip and precast slab gels.
Approx. gel size
(w × l, cm)
Number of
gels
Gel thickness
(mm)
IPG strip length
(cm)
Total separation time.
(h:m)
Vertical .
Ettan DALTsix*
26 × 20
1–6
1, 1.5
18, 24
4:00–6:30
Ettan DALTtwelve*
26 × 20
1–12
1, 1.5
18, 24
5:00–7:00
miniVE or SE 260
8 × 9.5
1–2
1, 1.5
7
1:30
SE 600 Ruby
14 × 16
16 × 16†
16 × 8§
1–4‡
1–4‡
1–4‡
1, 1.5
1, 1.5
1, 1.5
11
13
13
3:00–5:00
3:00–5:00
3:00–4:00
Flatbed .
Multiphor II
ExcelGel 2-D
Homogeneous 12.5*
24.5 × 11
1
0.5
all
1:45
ExcelGel Gradient
XL 12–14*
24.5 × 18
1
0.5
all
3:20
* Multiple shorter Immobiline DryStrip gels (two 11-cm strips or three 7-cm strips) fit on one gel.
If 1-cm-wide spacers are used.
†
An accessory divider plate increases the capacity to four gels.
‡
Up to eight mini-format separations can be simultaneously achieved using the shorter (8 cm) glass plates combined with divider plates.
§
80-6429-60 AD 13
Vertical systems
Vertical systems offer relative ease of use and the possibility of performing multiple separations simultaneously.
Vertical 2-D gels can be either 1 or 1.5 mm thick.
Ettan DALTsix (Fig 2) allows intermediate throughput of up to six high-resolution second-dimension gels. The unit
accommodates 18- or 24-cm Immobiline DryStrip gels that can be used with either precast or lab-cast large-format
Ettan DALT gels. A pump mounted under the lower chamber recirculates buffer around the cassettes for efficient
temperature regulation (in conjunction with MultiTemp III Thermostatic Circulator).
For maximal resolution, reproducibility, and capacity, the large-gel format of the Ettan DALTtwelve system (Fig 3) is
recommended. Precast large-format Ettan DALT gels on plastic film supports offer the convenience of ready-to-use
gels. The system can accommodate the entire length of an 18- or 24-cm Immobiline DryStrip gel (plus molecular
weight markers), and up to 12 gels can be run simultaneously. Integrated Peltier temperature control and a buffer
circulation pump provide a precise and uniform thermal environment. Up to fourteen 1-mm-thick gels can be cast
simultaneously in the Ettan DALTtwelve Gel Caster.
For rapid results, the mini-gel units—miniVE (Fig 4) or SE 260 (Fig 5)—are recommended. The second-dimension
separation is typically complete in 1–2 h. The use of mini-gels for the second dimension is ideal when quick profiling is
required or when the protein pattern is relatively simple.
For increased throughput and resolution, the standard-sized SE 600 Ruby Vertical Electrophoresis System (Fig 6) is
recommended. SE 600 Ruby accommodates up to four 16-cm-long gels and the built-in heat exchanger offers cooling
capability (in conjunction with MultiTemp III Thermostatic Circulator) for increased reproducibility. The standard spacer
width is 2 cm, giving a 14-cm-wide gel. If additional space for molecular weight markers is desired at both ends of a
13-cm Immobiline DryStrip gel, 1-cm-wide spacers are available for the preparation of 16-cm-wide gels. Short 8-cm
clamps, plates, and spacers are available for preparing gels that are 14–16 cm wide and 8 cm long. These short gels
may be used for rapid, simultaneous second-dimensional analysis of many 7-cm Immobiline DryStrip gels.
Multiphor II Electrophoresis System
The flatbed Multiphor II Electrophoresis System (Fig 7) provides excellent resolution and relatively rapid separations in
a large-format gel. Precast ExcelGel products offer the convenience of ready-to-use gels and buffer strips.
The protein loading capacity of an Immobiline DryStrip gel can exceed the capacity of the thin, horizontal,
second-dimension gel, so thicker vertical second-dimension gels are preferred for micropreparative
separations.
The Multiphor Electrophoresis System is not recommended for the second-dimension step if pH 6–9, 6–11, or
7–11 NL Immobiline DryStrip gels have been used for the first-dimension separation.
14 80-6429-60 AD
Good laboratory practice
Always wear gloves when handling Immobiline DryStrip gels, SDS polyacrylamide gels, ExcelGel Buffer Strips,
and any equipment that these items will contact. The use of gloves will reduce protein contamination that can
produce spurious spots or bands in 2-D patterns.
Clean all assemblies that will be in contact with the gels or samples using a detergent designed for glassware,
and rinse well with distilled water. This is particularly important when highly sensitive mass spectrometry
techniques are employed for spot identification and characterization. A special detergent is available for the
Strip Holders and Manifold (see chapter 2).
Always use the highest quality reagents and the purest water available.
Some of the chemicals used in the procedures—acrylamide, N,N’-methylenebisacrylamide, ammonium
persulfate, TEMED, thiourea, DTT, iodoacetamide, and DeStreak™ Reagent—are very hazardous. Acrylamide
monomer, for example, is a neurotoxin and suspected carcinogen. Read the manufacturer’s safety data sheet
(MSDS) detailing the properties and precautions for all chemicals in your laboratory. These safety data sheets
should be reviewed prior to starting the procedures described in this handbook. General handling procedures
for hazardous chemicals include using double latex gloves for all protocols. Hazardous materials should
be weighed in a fume hood while wearing a disposable dust mask. Follow all local rules and regulations for
handling and disposal of materials.
80-6429-60 AD 15
16 80-6429-60 AD
1. Sample preparation
1.0 General strategy
Appropriate sample preparation is absolutely essential for good 2-D electrophoresis results. Due to the great diversity
of protein sample types and origins, the optimal sample preparation procedure for any given sample must be
determined empirically. Ideally, the process will result in the complete solubilization, disaggregation, denaturation, and
reduction of the proteins in the sample.
There are several important differences in sample preparation for 2-D Fluorescence Difference Gel Electrophoresis
(2-D DIGE). See section 6.3.2 for more information.
When developing a sample preparation strategy, it is important to have a clear idea of what is desired in the final 2-D
result. Is the goal to view as many proteins as possible, or is only a subset of the proteins in the sample of potential
interest? Which is more important—complete sample representation or a clear, reproducible pattern? Additional
sample preparation steps can improve the quality of the final result, but each additional step can result in the
selective loss of protein species. The trade-off between improved sample quality and complete protein representation
must therefore be carefully considered.
In order to characterize specific proteins in a complex protein mixture, the proteins of interest must be completely
soluble under electrophoresis conditions. Different treatments and conditions are required to solubilize different
types of protein samples; some proteins are naturally found in complexes with membranes, nucleic acids, or other
proteins, some proteins form various nonspecific aggregates, and some proteins precipitate when removed from their
normal environment. The effectiveness of solubilization depends on the choice of cell disruption method, protein
concentration and dissolution method, choice of detergents, and composition of the sample solution. If any of these steps
are not optimized for a particular sample, separations may be incomplete or distorted and information may be lost.
1.0.1 Cell disruption, protection from proteolysis, fractionation
To fully analyze all intracellular proteins, the cells must be effectively disrupted. Choice of disruption method depends on
whether the sample is derived from cell suspensions, solid tissue, or other biological material and whether the analysis
is targeting all proteins or just a particular subcellular fraction. Gentle and vigorous lysis methods are discussed in
sections 1.1.1 and 1.1.2, respectively. A protocol for grinding cells using Sample Grinding Kit can be found in section 1.1.3.
Proteases may be liberated upon cell disruption. Proteolysis greatly complicates analysis of the 2-D gel result, thus
the protein sample should be protected from proteolysis during cell disruption and subsequent preparation. Protease
inhibition is discussed in section 1.2. Section 1.2.1 provides protocols for use of Protease Inhibitor Mix.
If only a subset of the proteins in a tissue or cell type is of interest, fractionation can be employed during sample
preparation. If proteins from one particular subcellular compartment (e.g. nuclei, mitochondria, plasma membrane)
are desired, the organelle of interest can be purified by differential centrifugation or other means prior to solubilization
of proteins for 2-D electrophoresis. The sample can also be fractionated by solubility under different extraction
conditions prior to 2-D electrophoresis (see references 9–13 for experimental conditions).
1.0.2 Precipitation and removal of interfering substances
In whole cell lysates, proteins are present in a wide dynamic range of concentrations. In such a situation, abundant
proteins may mask identification of less abundant proteins of interest. An effective proteome analysis will naturally
require separation of abundant proteins and enrichment of low-abundance proteins to bring the latter into detectable
range. This allows for improved resolution when an individual fraction is analyzed, provides less crowded 2-D maps,
simplifies analysis and interpretation, and increases the chances of discovering novel proteins of diagnostic or
therapeutic interest.
80-6429-60 AD 17
Precipitation of the proteins in the sample and removal of interfering substances are optional steps. The decision to
employ these steps depends on the nature of the sample and the experimental goal. Precipitation procedures, which
are used both to concentrate the sample and to separate the proteins from potentially interfering substances, are
described in section 1.4. Sections 1.4.1 provides protocols for sample clean-up using 2-D Clean-Up Kit.
Section 1.5 discusses the effects that contaminants (salts, small ionic molecules, albumin and IgG in human serum,
ionic detergents, nucleic acids, polysaccharides, lipids, and phenolic compounds) might have on the 2-D result if
they are not removed; the section also discusses removal techniques that eliminate specific contaminants from
the sample. Protocols are provided for desalting using Mini Dialysis Kit (section 1.5.1), removing undesirable nucleic
acids using Nuclease Mix (section 1.5.2), and eliminating problems associated with the presence of albumin and
immunoglobulin G (IgG) from human plasma using Albumin and IgG Removal Kit (section 1.5.3).
In general, it is advisable to keep sample preparation as simple as possible. A sample with low protein concentration
and a high salt concentration, for example, could be desalted then concentrated by lyophilization, or precipitated
with TCA and ice-cold acetone and resolubilized with rehydration solution. In some instances the option of simply
diluting the sample with rehydration solution may be sufficient. If problems with protein concentration or interfering
substances are otherwise insurmountable, then precipitation or contaminant removal steps may be necessary.
1.0.3 Additional aspects of sample preparation
The composition of the sample solution is particularly critical for 2-D electrophoresis because solubilization treatments
for the first-dimension separation must not affect the protein pI, or leave the sample in a highly conductive solution.
In general, concentrated urea (or combinations of urea and thiourea) and one or more detergents are used. Sample
solution composition is discussed in section 1.6.
Accurate quantitation of protein in samples prepared for electrophoresis can be difficult because many of the
reagents used to prepare and solubilize samples for electrophoresis (e.g. chaotropes, carrier ampholytes, detergents,
and reductants) are incompatible with common protein assays. Section 1.7 discusses this topic. Section 1.7.1 provides
a protocol for using 2-D Quant Kit to overcome this problem.
The above-mentioned sample preparation kits from GE Healthcare simplify preparation procedures and improve
sample quality, which is essential for obtaining good electrophoresis results. Table 4 summarizes the kits available;
these kits are described in more detail in following sections of this chapter.
Table 4. Sample Preparation Kits.
Product
Quantity
Use
Sample Grinding Kit
50 samples disrupts up to 100 mg tissue or cell sample
Protease Inhibitor Mix
1 ml
inhibits proteases
2-D Clean-Up Kit
50 samples
removes interfering material 1–100 µl
Mini Dialysis Kit
50 samples
1 kDa cut-off, up to 250 µl
Mini Dialysis Kit
50 samples
1 kDa cut-off, up to 2 ml
Mini Dialysis Kit
50 samples
8 kDa cut-off, up to 250 µl
Mini Dialysis Kit
50 samples
8 kDa cut-off, up to 2 ml
Nuclease Mix
0.5 ml
removes nucleic acids
Albumin and IgG Removal Kit
10 samples
removes albumin and IgG from human serum
2-D Quant Kit
500 assays
quantitation of 1–50 µl, up to 50 µg protein
Vivaspin
25 pack
exchanges buffer and concentrates sample
2-D Protein Extraction Buffer Trial Kit
6 × for 10 ml
prepares high quality protein lysates
illustra™ triplePrep Kit
50 preps
isolates simultaneously DNA, RNA and protein from the same sample
18 80-6429-60 AD
1.0.4 General sample preparation guidelines
Keep the sample preparation strategy as simple as possible to avoid protein losses. Additional sample
preparation steps may improve the quality of the final 2-D result, but at the possible expense of selective protein
loss.
Perform a literature search to determine if others have already worked out a sample preparation strategy.
Discussion groups such as the one at www.gehealthcare.com can also be helpful.
The cells or tissue should be disrupted in such a way as to minimize proteolysis and other types of protein
degradation. Cell disruption should be performed at as low a temperature as possible and with a minimum
of heat generation. Cell disruption should ideally be carried out directly into a strongly denaturing solution
containing protease inhibitors.
Preserve sample quality by preparing the sample just prior to IEF or storing samples in aliquots at -40 °C or
below. Do not expose samples to repeated freezing and thawing.
Remove all particulate material by ultracentrifugation. Solid particles and lipids must be removed because
they will block the pores in the electrophoresis gel.
To avoid modification of proteins, never heat a sample after adding urea. If the sample contains urea, the
solution temperature must not exceed 37 °C. Elevated temperatures cause urea to hydrolyze to isocyanate,
which modifies proteins by carbamylation, resulting in artifactual “charge trains.”
This chapter describes methods of sample preparation for 2-D electrophoresis using precast Immobiline
DryStrip gels available from GE Healthcare. Optimal protein loads for Immobiline DryStrip gels are discussed in
section 2.5. For more information on using Immobiline DryStrip gels and related equipment for IEF and
2-D electrophoresis, see chapters 2 and 3. For more specific guidance on preparing samples for application to
Immobiline DryStrip gels, see references 14–16.
80-6429-60 AD 19
1.1 Methods of cell disruption
Tables 5 and 6 list some standard mechanical and chemical disruption methods. Cell disruption should be performed at
low temperature; keep the sample on ice as much as possible and use chilled solutions.
Proteases may be liberated upon cell disruption, thus the protein sample should be protected from proteolysis if one
of the methods described in this section is to be used. It is generally preferable to disrupt the sample material directly
into a strongly denaturing lysis solution to rapidly inactivate proteases and other enzymatic activities that may modify
proteins. Cell disruption is often carried out in an appropriate solubilization solution for the proteins of interest (see
references 17 and 18 for general information on tissue disruption and cell lysis).
1.1.1 Gentle lysis methods
Gentle lysis methods are generally employed when the sample of interest consists of easily lysed cells (such as tissue
culture cells, blood cells, and some microorganisms). Gentle lysis methods can also be employed when only one particular
subcellular fraction is to be analyzed. For example, conditions can be chosen in which only cytoplasmic proteins are
released, or intact mitochondria or other organelles are recovered by differential centrifugation. Sometimes these
techniques are combined (e.g. osmotic lysis following enzymatic treatment, freeze-thaw in the presence of detergent).
Table 5 summarizes various options for gentle lysis.
Table 5. Gentle lysis methods.
Cell disruption method
Application
General procedure
Osmotic lysis (19).
This very gentle method is well-suited for
applications in which the lysate is to be
subsequently fractionated into subcellular
components.
Blood cells, tissue culture
cells
Suspend cells in a hypo-osmotic solution.
Bacterial cells,
tissue culture
cells
Rapidly freeze cell suspension using
liquid nitrogen, then thaw.
Repeat if necessary.
Freeze-thaw lysis (9, 17, 20).
Many types of cells can be lysed by
subjecting them to one or more cycles
of quick freezing and subsequent thawing.
Detergent lysis (21, 22).
Detergents solubilize cellular membranes,
Tissue culture
lysing cells and liberating their contents.
cells
Suspend cells in lysis solution containing detergent.
Cells can often be lysed directly into sample solution
or rehydration solution because these solutions
always contain detergent. See appendix I, solution A
for an example of a widely used lysis solution.
Further examples of this technique are given in
references 21 and 22.
If an anionic detergent such as SDS is used for lysis,
one of the following preparation steps is required to
ensure that the SDS will not interfere with IEF:
• Dilute the lysed sample with a solution containing
an excess of nonionic or zwitterionic detergent
OR
• Separate the SDS from the sample protein by
acetone precipitation.
Enzymatic lysis (23, 24).
Cells with cell walls can be lysed gently
following enzymatic removal of the cell wall.
This must be done with an enzyme specific
for the type of cell to be lysed (e.g. lysozyme
for bacterial cells, cellulase and pectinase
for plant cells, lyticase for yeast cells).
20 80-6429-60 AD
Plant tissue,
bacterial cells,
fungal cells
Treat cells with enzyme in an iso-osmotic solution.
1.1.2 More vigorous lysis methods
These methods are employed when cells are less easily disrupted, i.e. cells in solid tissues or cells with tough cell walls.
More vigorous lysis methods will result in complete disruption of the cells, but care must be taken to avoid heating or
foaming during these procedures. Table 6 summarizes these options.
Table 6. More vigorous lysis methods.
Cell disruption method
Application
Sonication (5, 25, 26).
Ultrasonic waves generated by a sonicator
Cell suspensions
lyse cells through shear forces.
Complete shearing is obtained when maximal agitation is achieved, but care
must be taken to minimize heating
and foaming.
French pressure cell (23, 24, 27).
Cells are lysed by shear forces resulting
from forcing suspension through a small
orifice under high pressure.
Microorganisms with cell walls (bacteria, algae,
yeasts)
Grinding (5, 8, 28, 29).
Some cell types can be lysed by grinding
Solid tissues,
with a mortar and pestle.
microorganisms
Mechanical homogenization (9, 19, 30–32).
Solid tissues
Many different devices can be used to
mechanically homogenize tissues.
Hand-held devices such as Dounce or
Potter-Elvehjem homogenizers can be used
to disrupt cell suspensions or relatively soft
tissues. Blenders, or other motorized
devices,can be used for larger samples.
Homogenization is rapid and causes little
damage to proteins except from the proteases
that may be liberated upon disruption.
Glass bead homogenization (23, 24, 33).
The abrasive action of the vortexed Cell suspensions,
beads breaks cell walls, liberating the microorganisms
cellular contents.
General procedure
Sonicate cell suspension in short bursts to
avoid heating.
Cool on ice between bursts.
Place cell suspension in chilled French pressure cell.
Apply pressure and collect extruded lysate.
Tissue or cells are normally frozen with liquid
nitrogen and ground to a fine powder.
Alumina (Al2O3) or sand may aid grinding.
Chop tissue into small pieces if necessary.
Add chilled homogenization buffer (5–20 volumes
to volume of tissue). Homogenize briefly.
Clarify lysate by filtration and/or centrifugation.
Suspend cells in an equal volume of chilled lysis
solution and place into a sturdy tube. Add 1–3 g
of chilled glass beads per gram of wet cells.
Vortex for 1 min and incubate cells on ice 1 min.
Repeat vortexing and chilling two to four times.
1.1.3 Processing small tissue or cell samples using Sample Grinding Kit
Sample Grinding Kit is designed to disrupt cell or tissue samples. It utilizes an abrasive grinding resin and grinding pestle
to rupture cells for protein extraction. Intracellular organelles are also disrupted, resulting in the liberation and extraction
of all proteins soluble in the extraction solution. Samples of 100 mg or less can be processed in as little as 10 min.
The kit contains fifty 1.5-ml microcentrifuge tubes, each containing a small quantity of abrasive grinding resin suspended
in water. The tube is centrifuged to pellet the resin and the water is removed. The methodology is outlined in Figure 8.
sample
grinding pestle
a
b
c
d
Fig 8. Schematic of the method used in the Sample Grinding Kit. (a) Pellet grinding resin in microcentrifuge tube. (b) Add sample and
extraction solution. Disrupt sample by grinding with pestle. (c) Centrifuge to separate cellular debris and resin. (d) Collect supernatant.
80-6429-60 AD 21
The extraction solution of choice is added to the tube along with the sample to be ground. A disposable pestle is
supplied to grind the sample. Immediately after grinding, cellular debris and grinding resin are removed by 5–10 min of
centrifugation. If desired following extraction, the sample solution may be treated to remove interfering substances
using 2-D Clean-Up Kit (see section 1.4.1).
Protocol: Sample Grinding Kit
Components supplied
Microcentrifuge grinding tubes containing grinding resin suspended in water, disposable pestles for sample grinding.
Required but not provided
Microcentrifuge capable of at least 12000 x g, vortex mixer, extraction solution.
Preliminary notes
Samples can be extracted into 8 M urea and 4% CHAPS, or into 7 M urea, 2 M thiourea, and 4% CHAPS (see solutions
A and B in appendix I). Alternative nonionic detergents or protease inhibitors can be added during extraction. Carrier
ampholytes (Pharmalyte™ reagents, Ampholines, or IPG Buffers) can be added at concentrations up to
2% for standard protocols but should not be added during protein extraction for labeling in 2-D DIGE.
1. Briefly centrifuge the grinding resin at maximum speed in the 1.5-ml microcentrifuge tubes provided in the kit
(Fig 8A). Remove supernatant with micropipette.
2. Add sample (up to 100 mg) and extraction solution of choice (200–300 µl) (see appendix I, solutions A and B).
Tissue can be cut up with a scalpel or frozen with liquid nitrogen and broken with mortar and pestle to
yield tissue fragments. Cell suspensions can be centrifuged with the grinding resin and resuspended in
extraction solution.
3. Grind sample thoroughly (up to 1 min) with the disposable pestle included in the kit (Fig 8B).
4. Limit extraction solution to 200–300 µl during grinding to prevent liquid from splashing out of the tube. Additional
extraction solution may be added to the tube following grinding (up to 1 ml).
5. Separate resin and debris by centrifugation for 5–10 min at maximum speed (Fig 8C).
6. Collect the supernatant and transfer to another tube (Fig 8D). If desired, proceed with further clean-up steps
using 2-D Clean-Up Kit (section 1.4.1).
1.1.4 Preparing samples from “difficult” protein sources
To prepare proteins from tissues that are dilute sources of protein and contain high levels of interfering substances
(e.g. plant tissues), the following procedure is recommended. This method produces protein solutions substantially free
of salts, nucleic acids, and other contaminants:
1. Grind tissue in mortar and pestle with liquid nitrogen.
2. Suspend powder in 10% TCA with 0.3% DTT in acetone.
3. Keep at -18 ºC overnight and centrifuge. Wash pellet with acetone.
4. Dry and resuspend in 9 M urea, 2% CHAPS, 1% DTT, 2% Pharmalyte 3–10 (52, 64).
Samples should remain in sample solution at room temperature for at least 30 min for full denaturation and
solubilization prior to centrifugation and subsequent sample application. Heating of the sample in the presence of
detergent can aid solubilization, but should only be done prior to the addition of urea. Sonication helps speed up
solubilization, particularly from material that is otherwise difficult to resuspend.
22 80-6429-60 AD
1.2 Protecting against proteolysis
When cells are lysed, proteases are often liberated or activated. Degradation of proteins through protease action
greatly complicates the analysis of 2-D electrophoresis results, so measures should be taken to avoid this problem. If
possible, inhibit proteases by disrupting the sample directly into strong denaturants such as 8 M urea, 10% TCA, or 2%
SDS (34–38). Proteases are less active at lower temperatures, so sample preparation should be carried out at as low a
temperature as possible. In addition, proteolysis can often be inhibited by preparing the sample in the presence of Tris,
sodium carbonate, or basic carrier ampholyte mixtures.
These approaches alone often provide sufficient protection against proteolysis. However, some proteases may retain
activity even under these conditions. In these cases, protease inhibitors may be used. Individual protease inhibitors are
only active against specific classes of proteases, so it is usually advisable to use a combination of protease inhibitors.
Broad-range protease inhibitor “cocktails” are available from a number of commercial sources. GE Healthcare offers
Protease Inhibitor Mix; see section 1.2.1 for more details and a description of the protocol.
Table 7 lists common protease inhibitors and the proteases they inhibit. For a more comprehensive discussion of
protease inhibition, see references 15, 31, and 39–43.
Table 7. Protease inhibitors.
Protease inhibitor
Effective against:
PMSF.
PMSF is an irreversible inhibitor (Phenylmethylsulfonyl fluoride)
Most commonly used inhibitor.
that inactivates:
Use at concentrations up to 1 mM. • serine proteases
• some cysteine proteases
AEBSF
(Aminoethyl benzylsulfonyl fluoride or Pefabloc™ SC Serine Protease Inhibitor)
Use at concentrations up to 4 mM.
AEBSF is similar to PMSF in
its inhibitory activity, but is
more soluble and less toxic.
Limitations
PMSF rapidly becomes inactive in aqueous solutions:
Prepare just prior to use.
PMSF may be less effective in the presence of thiol
reagents such as DTT or 2-mercaptoethanol. This
limitation can be overcome by disrupting the sample
into PMSF-containing solution lacking thiol reagents.
Thiol reagents can be added at a later stage.
PMSF is very toxic.
AEBSF-induced modifications can potentially
alter the pI of a protein.
EDTA or EGTA.
Use at 1 mM.
These compounds inhibit
metalloproteases by chelating free
metal ions required for activity.
Peptide protease inhibitors
(e.g. leupeptin, pepstatin, aprotinin, bestatin)
• reversible inhibitors
• active in the presence of DTT
• active at low concentrations under a variety of conditions
Use at 2–20 µg/ml.
Leupeptin inhibits many serine
Peptide protease inhibitors are:
and cysteine proteases.
• expensive.
Pepstatin inhibits aspartyl
• small peptides and thus may appear on the
proteases (e.g. acidic proteases 2-D map, depending on the size range separated
such as pepsin). Aprotinin inhibits by the second-dimension gel.
many serine proteases. Pepstatin does not inhibit any proteases that
Bestatin inhibits aminopeptidases.
are active at pH 9.
TLCK, TPCK
(Tosyl lysine chloromethyl ketone,
tosyl phenylalanine chloromethyl
ketone)
Use at 0.1–0.5 mM.
Benzamidine
Use at 1–3 mM.
These compounds irreversibly
inhibit many serine and
cysteine proteases.
Benzamidine inhibits serine proteases.
80-6429-60 AD 23
1.2.1 Protease inhibition using Protease Inhibitor Mix
Protease Inhibitor Mix from GE Healthcare contains an optimized concentration of competitive and noncompetitive
protease inhibitors that effectively inhibit serine, cysteine, metalloproteases, and calpain proteases. The kit is suitable
for the protection of proteins during purification from animal tissues, plant tissues, yeast, and bacteria.
Protocol: Protease Inhibitor Mix
Reagents supplied
Protease Inhibitor Mix (100× solution), 1 ml.
Required but not provided
Microcentrifuge, vortex mixer, extraction solution.
Preliminary notes
Protease Inhibitor Mix is provided free of EDTA as some proteins require divalent cations such as Ca2+, Mg2+, or Mn2+ for
their biological activity. In such circumstances, the presence of EDTA may be detrimental to sample protein activity.
Samples can be extracted into 8 M urea and 4% CHAPS, or into 7 M urea, 2 M thiourea, and 4% CHAPS (see solutions A
and B in appendix I). Alternative nonionic detergents or protease inhibitors can be added during extraction. Carrier
ampholytes (Pharmalytes, Ampholines, or IPG Buffers) can be added at concentrations up to 2% for standard
protocols but should not be added during protein extraction for labeling in 2-D DIGE.
1. Allow the solution to warm to room temperature.
2. Vortex briefly before using, as the solution is in suspension form.
3. Dilute Protease Inhibitor Mix 1:100 (10 µl/ml) in an appropriate volume of extraction buffer or extract.
Further options
• If a higher potency of protease inhibition is required, add Protease Inhibitor Mix at a concentration of 20–30 µl/ml
to give a 2–3× final concentration.
• For the inhibition of metalloproteases, add EDTA directly in an appropriate volume of extraction buffer or extract
to give a final concentration of 5 mM EDTA in the reaction.
EDTA must not be added if the solution is to be used in conjunction with Nuclease Mix, because EDTA acts as a
nuclease inhibitor.
1.3 Fractionation of protein lysates
Proteome studies involving quantitative comparisons of total cell protein profiles from two or more experimental
samples require methods for highly reproducible separation of cell or tissue protein extracts. 2-D gel electrophoresis is
currently the only proven method for simultaneous separation of highly complex protein mixtures and quantitative
comparison of changes in protein profiles of cells, tissues, or whole organisms.
Although 2-D electrophoresis gives the highest resolution of all available protein separation methods, a drawback
when complex protein lysates are run on 2-D gels without prefractionation is that the resulting 2-D gel is crowded
with spots, making interpretation of results difficult. Typically, a 2-D gel can yield anywhere between 1000 and
4000 spots under favorable conditions, but the presence of many of the most interesting proteins, particularly
low-abundance proteins, can be masked. Where high protein loads are employed, such as with preparative 2-D
gels, higher protein amount loaded onto the gel results in 2-D patterns having poorer resolution, with spots of very
abundant proteins overlaying the spots of less abundant proteins. If the loads are increased even more, abundant
proteins become predominant and the separation is poor.
Thus the greatest challenge in protein discovery and analysis of important proteins is the right sample preparation
strategy for 2-D electrophoresis. Strategies for prefractionation of samples for 2-D electrophoresis appear to be
the most promising approach for increasing the number of protein components that can be visualized in complex
proteomes such as mammalian cells, tissues, and physiological fluids. In addition, removal of contaminants is part of
the strategy. For this purpose, GE Healthcare provides the Albumin and IgG Removal Kit, which includes an affinity gel
to selectively remove albumin and IgG contaminants in human serum 2-D maps. The use of Albumin and IgG Removal
Kit to improve 2-D electrophoresis of human serum is described in detail in section 1.5.3.
24 80-6429-60 AD
1.4 Precipitation procedures
Protein precipitation is an optional step in sample preparation for 2-D electrophoresis. Precipitation, followed
by resuspension in sample solution, is generally employed to selectively separate proteins in the sample from
contaminating species such as salts, detergents, nucleic acids, lipids, etc., that would otherwise interfere with the 2-D
result. Precipitation followed by resuspension can also be employed to prepare a concentrated protein sample from
a dilute source (e.g. plant tissues, urine). Note, however, that no precipitation technique is completely efficient, and
some proteins may not readily resuspend following precipitation. Thus, employing a precipitation step during sample
preparation can alter the protein profile of a sample. When complete and accurate representation of all the proteins
in a sample is of paramount interest, precipitation and resuspension should be avoided.
2-D Clean-Up Kit from GE Healthcare can be used to remove contaminating substances and improve the 2-D
electrophoresis pattern. Proteins are precipitated with a combination of precipitation reagents while the interfering
substances, such as nucleic acids, salts, lipids, or detergents, remain in solution. Samples can be resuspended in the
desired denaturing solution for IEF. Each kit can process 50 samples of up to 100 µl each. Section 1.4.1 describes the
kit and provides a protocol for use.
Table 8 lists some of the precipitation techniques that can be used. If sample preparation requires precipitation,
typically only one precipitation technique is employed.
Table 8. Precipitation procedures.
Precipitation method
General procedure
Ammonium sulfate precipitation.
(“Salting out”)
Prepare protein so that the final concentration In the presence of high salt
of the protein solution is > 1 mg/ml in a buffer
concentrations, proteins tend to
solution that is > 50 mM and contains EDTA.
aggregate and precipitate out of
Slowly add ammonium sulfate to the desired
solution. Many potential
percent saturation (44) and stir for 10–30 min.
contaminants (e.g. nucleic acids)
Pellet proteins by centrifugation.
will remain in solution.
Limitations
Many proteins remain soluble at high
salt concentrations, so this method is
not recommended when total protein
representation is desired.
This method can, however, be used
for prefractionation or enrichment.
Residual ammonium sulfate will
interfere with IEF and must be
removed (45).
See section 1.5 on removal of salts.
TCA precipitation.
TCA (trichloroacetic acid) is a very
effective protein precipitant.
TCA is added to the extract to a final concentration of 10–20% and the proteins
are allowed to precipitate on ice for 30 min (46).
Alternatively, tissue may be homogenized
directly into 10–20% TCA (35, 47).
This approach limits proteolysis and
other protein modifications.
Centrifuge and wash pellet with acetone or ethanol to remove residual TCA.
Proteins may be difficult to resolubilize
and may not resolubilize completely.
Residual TCA must be removed by
extensive washing with acetone or
ethanol.
Extended exposure to this low
pH solution may cause some protein
degradation or modification.
Acetone precipitation
This organic solvent is commonly
used to precipitate proteins. Many organic-soluble contaminants
(e.g. detergents, lipids) will remain
in solution.
Add at least three volumes of ice-cold acetone
to the extract. Allow proteins to precipitate
at -20 ºC for at least 2 h. Pellet proteins by
centrifugation (46, 48–50). Residual acetone
is removed by air-drying or lyophilization.
Incomplete recovery of all proteins.
Precipitation with TCA in acetone
The combination of TCA and
acetone is commonly used to
precipitate proteins during sample
preparation for 2-D electrophoresis,
and is more effective than either
TCA or acetone alone.
Suspend lysed or disrupted sample in
10% TCA in acetone with either
0.07% 2-mercaptoethanol or 20 mM DTT.
Precipitate proteins for at least 45 min at
-20 ºC. Pellet proteins by centrifugation and
wash pellet with cold acetone containing either
0.07% 2-mercaptoethanol or 20 mM DTT.
Remove residual acetone by air drying or
lyophilization (5, 28, 34, 43, 51, 52).
Proteins may be difficult to resolubilize
and may not resolubilize completely.
Extended exposure to this low pH
solution may cause some protein
degradation or modification.
Compatibility of acetone with tubes
may be an issue.
continues on following page
80-6429-60 AD 25
Table 8. Precipitation procedures (continued).
Precipitation method
General procedure
Limitations
Precipitation with ammonium .
acetate in methanol following .
phenol extraction
This technique has proven useful
with plant samples containing high
levels of interfering substances.
Proteins in the sample are extracted into
water- or buffer-saturated phenol. Proteins
are precipitated from the phenol phase with
0.1 M ammonium acetate in methanol.
The pellet is washed several times with
ammonium acetate in methanol and then
with acetone.
Residual acetone is evaporated (42, 43, 47, 53).
The method is complicated and
time consuming.
For an overview of precipitation techniques, see references 17, 18, and 44.
1.4.1 Cleaning up samples using 2-D Clean-Up Kit
2-D Clean-Up Kit is designed to prepare samples that would otherwise produce poor 2-D results due to high
conductivity, high levels of interfering substances, or low concentration of protein.
Current methods of protein precipitation suffer from several significant disadvantages:
• Precipitation can be incomplete, resulting in the loss of proteins from the sample and introduction of
bias into the 2-D result.
• The precipitated protein can be difficult to resuspend and often cannot be fully recovered.
• The precipitation procedure can itself introduce ions that interfere with first-dimension IEF.
• Precipitation can be time-consuming, requiring overnight incubation of the sample.
2-D Clean-Up Kit circumvents these disadvantages by providing a method for selectively precipitating protein for
2-D electrophoresis. Protein can be quantitatively precipitated from a variety of sources without interference from detergents,
chaotropes, and other common reagents used to solubilize protein. Recovery is generally greater than 90%. The
procedure does not result in spot gain or loss, or changes in spot position relative to untreated samples. The precipitated
proteins are easily resuspended in 2-D sample solution. The procedure can be completed in less than one hour.
The overall quality of protein separation using 2-D Clean-Up Kit has been shown to be superior to that of samples
prepared by precipitation with acetone (54). Preparation of protein samples with the kit reduces horizontal streaking,
improves spot resolution, and increases the number of spots detected compared with samples treated by other
means (Fig 9 and Table 9).
Fig 9. 2-D Clean-Up Kit eliminates horizontal streaking caused by residual SDS. Sample: Rat liver extracted with 4% SDS, 40 mM Tris
base. First dimension: Approximately 20 µg rat liver protein, 7-cm Immobiline DryStrip pH 4–7, Ettan IPGphor Isoelectric Focusing System
17.5 kVh. Second dimension: SDS-PAGE (12.5%), SE 260 (8 × 9 cm gel). Stain: Silver Staining Kit, Protein.
26 80-6429-60 AD
Table 9. Effect of sample preparation on the number of protein spots detected in 2-D electrophoresis gels.
Sample preparation Number of silver-stained spots*
Protein extracted with urea buffer 726
Protein extracted with 1% Triton™ X-100 and precipitated with three volumes of acetone
758
Protein extracted with 1% Triton X-100 and purified using 2-D Clean-Up Kit
801
†
* Protein spots were detected using ImageMaster™ 2D Elite software.
9.8 M urea, 2% CHAPS, 0.5% IPG Buffer pH 3–10, 65 mM DTT.
†
The 2-D Clean-Up Kit procedure uses a combination of a unique precipitant and co-precipitant to quantitatively
precipitate the sample proteins while leaving interfering substances behind in the solution. The proteins are pelleted by
centrifugation and the precipitate is washed to further remove non-protein contaminants. The mixture is centrifuged
again and the resultant pellet can be easily resuspended into a 2-D sample solution of choice, compatible with firstdimension IEF.
The kit contains sufficient reagents to process 50 samples of up to 100 µl each. The procedure can be scaled-up for
larger volumes or more dilute samples.
Protocol: 2-D Clean-Up Kit
Reagents supplied
Precipitant, co-precipitant, wash buffer, wash additive.
Required but not provided
Ice bath, 1.5-ml capped microcentrifuge tubes, microcentrifuge capable of at least 12 000 × g, rehydration solution or
IEF sample solution for resuspension (see next section), vortex mixer.
Preliminary notes
Procedure A is applicable for sample volumes of 1–100 µl containing 1–100 µg of protein. For larger samples
containing more than 100 µg of protein, use procedure B.
Prior to starting the procedure, chill the wash buffer to -20 °C for at least 1 h.
A. For sample volumes of 1–100 µl (containing 1–100 µg of protein per sample)
Process the protein samples in 1.5-ml microcentrifuge tubes. All steps should be carried out on ice unless
otherwise specified.
1. Transfer 1–100 µl of protein sample (containing 1–100 µg protein) into a 1.5-ml microcentrifuge tube.
2. Add 300 µl of precipitant. Mix well by vortexing or inversion. Incubate the tube on ice (4–5 °C) for 15 min.
3. Add 300 µl of co-precipitant to the mixture of protein and precipitant. Mix by vortexing briefly.
4. Position the tubes in a microcentrifuge with cap-hinges facing outward. Centrifuge at maximum speed (at least
12 000 × g) for 5 min. Remove the tubes from the microcentrifuge as soon as centrifugation has finished.
A small pellet should be visible.
Proceed rapidly to the next step to avoid resuspension or dispersion of the pellet.
5. Remove as much of the supernatant as possible by decanting or careful pipetting. Do not disturb the pellet.
6. Carefully reposition the tubes in the microcentrifuge with the cap-hinges and pellets facing outward. Centrifuge
the tubes briefly to bring any remaining liquid to the bottom of the tubes. Use a pipette to remove the remaining
supernatant. There should be no visible liquid remaining in the tubes.
7. Without disturbing the pellet, layer 40 µl of co-precipitant on top of each pellet. Incubate the tubes on ice for 5 min.
8. Carefully reposition the tubes in the centrifuge with the cap-hinges facing outward. Centrifuge for 5 min. Use a
pipette to remove the supernatant.
9. Pipette 25 µl of distilled or deionized water on top of each pellet. Vortex each tube for 5–10 s. The pellet should
disperse, but not dissolve in the water.
80-6429-60 AD 27
10.Add 1 ml of wash buffer (prechilled for at least 1 h at -20 ºC) and 5 µl of wash additive to each tube. Vortex until
the pellets are fully dispersed.
Note: The protein pellet will not dissolve in the wash buffer.
11.Incubate the tubes at -20 °C for at least 30 min. Vortex for 20–30 s once every 10 min. At this stage, the tubes can
be stored at -20 ºC for up to one week with minimal protein degradation or modification.
12.Centrifuge the tubes at maximum speed (at least 12 000 × g) for 5 min.
13.Carefully remove and discard the supernatant. A white pellet should be visible. Allow the pellet to air dry briefly
(no more than 5 min).
Do not over-dry the pellet. If it becomes too dry, it will be difficult to resuspend.
14.Resuspend each pellet in an appropriate volume of rehydration or IEF sample loading solution for first-dimension IEF.
See next section for examples of rehydration solutions and volumes appropriate to different applications. Vortex
the tubes for at least 30 s. Incubate at room temperature. Vortex or aspirate and dispense using a pipette to
fully dissolve.
If the pellet is large or too dry, it may be difficult to resuspend fully. Sonication or treatment with the
Sample Grinding Kit (see section 1.1.3) can speed resuspension.
15.Centrifuge the tubes at maximum speed (at least 12 000 × g) for 5 min to remove any insoluble material and to
reduce any foam. The supernatant may be loaded directly onto first-dimension IEF or transferred to another tube
and stored at -80 ºC for later analysis.
B. For larger samples of more than 100 µg of protein
All steps should be carried out on ice unless otherwise specified.
1. Transfer the protein samples into tubes that can be centrifuged at 8000 × g. Each tube must have a capacity at
least 12-fold greater than the volume of the sample. Use only polypropylene, polyallomer, or glass tubes.
The wash buffer used later in the procedure is not compatible with many plastics. This limits the choice of
centrifuge tube materials.
2. For each volume of sample, add three volumes of precipitant. Mix well by vortexing or inversion. Incubate on ice
(4–5 °C) for 15 min.
3. For each original volume of sample, add three volumes of co-precipitant to the mixture of protein and precipitant.
Mix by vortexing briefly.
4. Position the tubes in a microcentrifuge with the cap-hinges facing outward. Centrifuge at 8000 × g for 10 min.
Remove the tubes from the microcentrifuge as soon as centrifugation has finished.
A pellet should be visible.
Proceed rapidly to the next step to avoid resuspension or diffusion of the pellet.
5. Remove as much of the supernatant as possible by decanting or careful pipetting. Do not disturb the pellet.
6. Carefully position the tubes in the microcentrifuge with the cap-hinges and pellets facing outward. Centrifuge the
tubes for at least 1 min to bring any remaining liquid to the bottom of the tubes. Use a pipette to remove the
remaining supernatant. There should be no visible liquid remaining in the tubes.
7. To each tube, add three-fold to four-fold more co-precipitant than the size of the pellet.
8. Carefully reposition the tubes in the microcentrifuge with the cap-hinges facing outward. Centrifuge for 5 min.
Use a pipette to remove the supernatant.
9. Pipette enough distilled or deionized water on top of each pellet to cover the pellet. Vortex each tube for several
seconds. The pellets should disperse, but not dissolve in the water.
10.Add 1 ml of wash buffer, prechilled for at least 1 h at -20 ºC to each tube. (For an initial sample volume of
0.1–0.3 ml, add 1 ml of wash buffer. However, the volume of wash buffer must be at least 10-fold greater than
the distilled/deionized water added in step 9.) Add 5 µl wash additive (use only 5 µl wash additive, regardless of
the original sample volume). Vortex until the pellet is fully dispersed.
Note: The protein pellet will not dissolve in the wash buffer.
11.Incubate the tubes at -20 °C for at least 30 min. Vortex for 20–30 s once every 10 min.
At this stage, the tubes can be stored at -20 ºC for up to one week with minimal protein degradation or modification.
12.Centrifuge the tubes at 8000 × g for 10 min.
28 80-6429-60 AD
13.Carefully remove and discard the supernatant. A white pellet should be visible. Allow the pellet to air dry briefly
(no more than 5 min).
Do not over-dry the pellet. If it becomes too dry, it will be difficult to resuspend.
14.Resuspend each pellet in rehydration solution for first-dimension IEF. The volume of rehydration solution used
can be as little as 1/20 of the volume of the original sample. See next section for examples of rehydration solutions
and volumes appropriate for different applications. Vortex the tube for 30 s. Incubate at room temperature.
Vortex or aspirate and dispense using a pipette to fully dissolve.
If the pellet is large or too dry, it may be difficult to resuspend fully. Sonication can speed resuspension.
15.Centrifuge the tubes at 8000 × g for 10 min to remove any insoluble material and to reduce any foam.
The supernatant may be loaded directly onto first-dimension IEF or transferred to another tube and stored
at -80 ºC for later analysis.
1.4.2 Resuspension of pellet
2-D Clean-Up Kit produces a protein pellet. When using cup loading, resuspend the pellet in sample preparation
solution (see appendix I). When using rehydration loading, resuspend the pellet in rehydration solution (see options 1
and 2 below), which is applied directly to the Immobiline DryStrip gel.
1. Rehydration solution containing 8 M urea
Use solution C in appendix I. This all-purpose solution gives clean, sharp 2-D separations.
2. Rehydration solution containing 7 M urea and 2 M thiourea
Use solution D in appendix I. This is a more strongly solubilizing solution that results in more spots in the final
2-D pattern.
Any other components added to the rehydration solution must either be uncharged or present at a concentration
of less than 5 mM. The addition of salts, acids, bases, and buffers is not recommended.
3. DeStreak Reagent
Use for basic strips. See section 2.6.2 for details on the reagent.
Sample resuspension volumes
The volume of rehydration solution used to resuspend the sample depends on the sample loading method and the
length of the Immobiline DryStrip gel used for the first-dimension separation. If using Ettan IPGphor 3 and the sample is
to be loaded onto the Immobiline DryStrip gel using a sample cup, the sample volume should not exceed 150 µl. If the
sample is to be loaded onto the Immobiline DryStrip gel by rehydration, the sample volumes shown in Table 10 should
be used according to the length of the Immobiline DryStrip gel.
Table 10. Sample volumes for different Immobiline DryStrip gel lengths.
Immobiline DryStrip gel length (cm)
Sample volume applied (µl)
7
125
11
200
13
250
18
340
24
450
The optimal quantity of protein to load varies widely depending on factors such as sample complexity, the length and
pH range of the Immobiline DryStrip gel, and the method of visualizing the 2-D gel separation. General guidelines are
given in chapter 2.
The protein concentration of the sample is best determined using the 2-D Quant Kit, which can accurately quantitate
protein in the presence of detergents, reductants, and other reagents used in sample preparation. See section 1.7
for details.
80-6429-60 AD 29
1.5 Other methods for removing contaminants
The first-dimension IEF step of 2-D electrophoresis is particularly sensitive to low-molecular-weight ionic impurities.
Non-protein impurities in the sample can interfere with separation and subsequent visualization of the 2-D gel
result, so sample preparation may require steps to rid the sample of these substances. Table 11 lists contaminants
that affect 2-D results and techniques for their removal. Reference 9 provides further discussion on the removal of
interfering substances. Mini Dialysis Kit, Albumin and IgG Removal Kit, and Nuclease Mix may be used to remove
interfering substances that affect 2-D results. Refer to section 1.4.1 for a discussion of 2-D Clean-Up Kit, which
selectively precipitates protein for 2-D analysis.
Salt contamination is the most frequent cause of insufficient focusing of protein spots.
Table 11. Contaminants that affect 2-D results.
Contaminant
Reason for removal
Removal techniques
Salts, residual buffers,
and other charged small
molecules that carry over
from sample preparation.
Salts disturb the electrophoresis process
and must be removed or maintained at
as low a concentration as possible.
Salts in the IPG strip result in high strip
conductivity. Focusing of the proteins will
not occur until the ions have moved to the
ends of the strips, prolonging the time
required for IEF. Water movement can also
occur, causing one end of the strip to dry
out and the other end to swell. Salt in the IPG strip can result in large regions at
either end of the IPG strip where proteins
do not focus (seen as horizontal streaking
or empty regions in the final result).
If the sample is rehydrated into the IPG
strip, the salt concentration in the
rehydration solution should be lower
than 10 mM.
If the sample is applied in sample cups,
salt concentrations of up to 50 mM in the
sample may be tolerated; however, proteins
may precipitate at the sample application
point as they abruptly move into a lower
salt environment.
Desalting can be performed by:
• dialysis
• spin dialysis
• gel filtration
• precipitation/resuspension
Dialysis is a very effective method for salt
removal resulting in minimal sample loss.
However, the process is time-consuming
and requires large volumes of solution.
Spin dialysis is quicker, but protein
adsorption onto the dialysis membrane
may be a problem. Spin dialysis should
be applied to samples prior to the addition
of urea and detergent.
Gel filtration can be acceptable but often
results in protein losses.
Precipitation/resuspension is an effective
means for removing salts and other
contaminants, but can also result in protein
losses (see section 1.4).
Endogenous small ionic
molecules (nucleotides,
metabolites,
phospholipids, etc).
Endogenous small ionic molecules are
present in any cell lysate.
These substances are often negatively
charged and can result in poor focusing
toward the anode.
TCA/acetone precipitation is particularly
effective at removing this sort of contaminant.
Other desalting techniques may be applied
(see above).
Albumin and IgG
in human serum
These two major protein components
of serum represent greater than 60% of the total protein in human serum content.
During gel analysis of serum, the high
concentration of albumin and IgG often
masks the presence of other proteins with
similar isoelectric point and/or molecular
weight. Therefore, removal of albumin and
IgG from serum samples, prior to electrophoresis, improves the resolution of lowerabundance proteins in two ways: by enabling
visualization of proteins that co-migrate with
albumin and IgG; and by removal of a large
portion of the total serum protein, which
allows an increase in the protein load of the
low-abundant proteins.
Affinity resins selectively remove these
contaminants from human serum.
continues on following page
30 80-6429-60 AD
Table 11. Contaminants that affect 2-D results (continued).
Contaminant
Reason for removal
Removal techniques
Ionic detergent
Ionic detergent (usually SDS) is often used
during protein extraction and solubilization,
but can strongly interfere with IEF.
SDS forms complexes with proteins,
and the resulting negatively charged
complex will not focus unless the SDS
is removed or sequestered.
Dilute the SDS-containing sample into a
rehydration solution containing a zwitterionic
or nonionic detergent (CHAPS, Triton X-100,
or Nonidet™ P-40 [NP-40]) so the final
concentration of SDS is 0.25% or lower and
of SDS is 0.25% or lower and the ratio of the
other detergent to SDS is at least 8:1 (27).
Acetone precipitation of the protein will
partially remove SDS.
Precipitation at room temperature will maximize
removal of SDS, but protein precipitation is
more complete at -20 °C (45).
Nucleic acids
Nucleic acids increase sample viscosity
and cause background smears.
(DNA, RNA)
High-molecular-weight nucleic acids can
clog gel pores.
Nucleic acids can bind to proteins through
electrostatic interactions, preventing
focusing.
If the separated sample proteins are
visualized by silver staining, nucleic acids
present in the gel will also stain, resulting
in a background smear on the 2-D gel.
Treat samples rich in nucleic acids with a
protease-free DNase/RNase mixture to reduce
the nucleic acids to mono- and oligonucleotides.
This is often done by adding 0.1 times the
volume of a solution containing 1 mg/ml DNase I,
0.25 mg/ml RNase A, and 50 mM MgCl2
followed by incubation on ice (33, 50).
Note: The DNase and RNase proteins may
appear on the 2-D map.
Ultracentrifugation can be used to remove
large nucleic acids; however, this technique
may also remove high-molecular-weight
proteins from the sample.
When using low ionic strength extraction
conditions, negatively charged nucleic acids
may form complexes with positively charged
proteins. High ionic strength extraction and/or
high-pH extraction may minimize these
interactions. (Note that salts added during
extraction must be subsequently removed;
see above.)
Polysaccharides
Polysaccharides can clog gel pores
causing either precipitation or extended
focusing times, resulting in horizontal streaking.
Some polysaccharides contain negative
charges and can complex with proteins
by electrostatic interactions.
Precipitate the sample in TCA, ammonium
sulfate, or phenol/ammonium acetate,
then centrifuge.
Ultracentrifugation will remove highmolecular-weight polysaccharides.
Employing the same methods used for
preventing protein-nucleic acid interactions may also be helpful (solubilize sample in SDS
or at high pH).
Lipids
Strongly denaturing conditions and detergents
minimize protein-lipid interactions.
Excess detergent may be necessary.
Precipitation with acetone removes some lipid.
Many proteins, particularly membrane
proteins, are complexed with lipids. This
reduces their solubility and can affect both
the pI and the molecular weight.
Lipids form complexes with detergents,
reducing the effectiveness of the detergent
as a protein-solubilizing agent.
When extracts of lipid-rich tissues are
centrifuged, there is often a lipid layer that
can be difficult to remove.
Phenolic compounds
Phenolic compounds are present in many
plant tissues and can modify proteins
through an enzyme-catalyzed oxidative
reaction (43, 49).
Prevent phenolic oxidation by employing
reductants during tissue extraction (e.g. DTT,
2-mercaptoethanol, sulfite, ascorbate).
Rapidly separate proteins from phenolic
compounds by precipitation techniques.
Inactivate polyphenol oxidase with inhibitors
such as diethyldithiocarbamic acid or thiourea.
Remove phenolic compounds by adsorption to
polyvinylpyrrolidone (PVP) or polyvinylpolypyrrolidone (PVPP).
continues on following page
80-6429-60 AD 31
Table 11. Contaminants that affect 2-D results (continued).
Contaminant
Reason for removal
Removal techniques
Insoluble material
Insoluble material in the sample can clog
gel pores and result in poor focusing.
Insoluble material is particularly problematic when the sample is applied using sample
cups as it can prevent protein entry into
the IPG strip.
Samples should always be clarified by
centrifugation prior to application in
first-dimension IEF.
Even relatively low concentrations of salts (< 5 mM) can slow down separation, prevent sharp focusing, or cause
disturbances that result in a poor-quality 2-D result. Low-molecular-weight ionic impurities can originate either as
endogenous components of the sample source or as salts and buffers introduced during preparation of the sample. In
either case, the ability of a sample to be effectively separated by first-dimension IEF, and the subsequent quality of the
2-D electrophoresis result can often be improved by dialyzing the sample prior to application. Mini Dialysis Kit is well
suited for this application because the capacity of the dialysis tubes (10–250 µl or 200–2000 µl) corresponds to typical
volume ranges for 2-D samples and because sample losses from the procedure are negligible.
1.5.1 Desalting samples using Mini Dialysis Kit
Mini Dialysis Kit is designed for the dialysis of small sample volumes with minimal handling and sample loss, offering a
simple solution to the handling problems of low-volume dialysis and reducing the pronounced streaking on 2-D gels
caused by low-molecular-weight contaminants (Fig 10). The kit contains dialysis tubes, each of which consists of a
sample tube with a cap that is fitted with a dialysis membrane. Sample is easily and quantitatively transferred into
and out of the tube by pipetting.
pH 3–10NL
pH 3–10NL
Undialyzed
Dialyzed with Mini Dialysis Kit
Fig 10. Effect of dialysis on 2-D resolution. Sample: E. coli protein extracted with 15 mM NaCl, 8 M urea, 0.5% Pharmalyte pH 3–10,
2% CHAPS. Dialysis: Mini Dialysis Kit 8 kDa, 250 µl, 17 h against 8 M urea. First dimension: Approximately 400 µg E. coli protein, 13-cm
Immobiline DryStrip pH 3–10 NL, Ettan IPGphor Isoelectric Focusing System 32 kVh. Second dimension: SDS-PAGE (12.5%), SE 600
vertical electrophoresis system (16 × 16 cm gel). Stain: Colloidal Coomassie™ G-250.
▼
dialysis membrane
▼
float
a
b
▼
c
d
Fig 11. Schematic of the method used in Mini Dialysis Kit. (a) Cap with dialysis membrane, conical inner sample tube. (b) Introduce
sample, screw on cap, and slide tube into float. (c) Invert and dialyze while stirring. (d) Spin briefly to collect sample.
The capped tube is inverted in a stirred beaker containing the solution against which the sample is to be dialyzed.
Salts and molecules smaller than the molecular weight cut-off of the dialysis membrane are effectively exchanged
through the membrane. Following dialysis, the tube is centrifuged briefly. This forces the entire contents of the dialysis
tube into the bottom of the tube, ensuring essentially 100% recovery. The dialyzing cap is replaced with a normal
32 80-6429-60 AD
cap for storage of the dialyzed sample (Fig 11). The kit is available with a choice of molecular weight cut-off (either
1 kDa or 8 kDa) and a choice of tubes for sample volumes of either 250 µl or 2 ml. Each kit contains dialysis tubes and
associated accessories sufficient for preparing 50 samples.
Dialysis times of a few hours to overnight are sufficient to reduce ionic contaminants to a level that does not interfere
with first-dimension IEF separation.
Since some detergents, notably Triton X-100 and SDS, form high-molecular-weight micelles at low concentration, they
cannot be effectively removed by dialysis. Other techniques, such as sample precipitation with 2-D Clean-Up Kit (see
section 1.4.1), must be used to remove these detergents.
Protocol: Mini Dialysis Kit
Components supplied
Dialysis tubes with caps incorporating a dialysis membrane (tubes for up to 250 µl or 2 ml sample are included in
the kit), caps (standard tube caps to seal the tubes following dialysis), floats (floating plastic sponges to suspend the
inverted dialysis tubes in the dialysis solution).
Required but not provided
Centrifuge (dependent on size of dialysis tube) capable of low speeds.
Preliminary notes
Prior to dialysis, samples for native IEF should be solubilized in water while samples for denaturing IEF for 2-D work should
be solubilized in a solution containing urea, reductant, and nonionic detergent. See sections 1.6.1 and 1.6.2 for details.
Handle dialysis tubes and caps with gloves.
Dialysis tubes are supplied in 0.05% (w/v) sodium azide and require rinsing before use.
1. Rinse dialysis tube and cap with distilled or deionized water (Fig 11A).
Keep cap covered with water until needed.
Do not allow cap with dialysis membrane to dry out.
2. Remove cap from water. Remove excess water with a micropipette. Ensure that the cap is tightly sealed.
3. Add sample to dialysis tube and replace dialysis cap (Fig 11B). For 250 µl dialysis tubes, use 10–250 µl of sample.
For 2-ml dialysis tubes, use 200 µl-2 ml of sample.
4. Invert dialysis tube ensuring that entire sample rests on dialysis membrane.
If the sample is viscous and does not initially rest on the membrane, the dialysis tube can be centrifuged in the
inverted position at 10–100 × g for no more than 6 s.
Spinning longer or faster may rupture the membrane.
5.
Secure dialysis tube to one of the floats provided. Place dialysis tube and float assembly (cap-end down) in a
beaker of the solution to be dialyzed against (e.g. water or 1% glycine for native IEF or sample buffer containing
urea, reducing agent, and nonionic detergent for denaturing IEF; see sections 1.6.1 and 1.6.2 for details).
Check that the dialysis membrane fully contacts the dialysis solution and that no large air bubbles are trapped
beneath the dialysis membrane. Remove any air bubbles by tilting the tube or squirting dialysis solution onto
the membrane.
6.
Dialyze while stirring (Fig 11C). During dialysis, invert dialysis tube to thoroughly mix contents.
Note: Optimal dialysis time depends on several factors, including the nature and volume of the sample, the
molecular weight cut-off of the dialysis membrane and the temperature. Normally, dialysis for 2 h to overnight is
sufficient to reduce ionic contaminants to a level that does not interfere with IEF separation. Dialysis may be
carried out at 4–8 °C to minimize sample degradation or modification, but this will slow down the dialysis.
Dialysis can be conducted at room temperature if degradation or modification is not a concern.
See section below on dialysis solutions.
7. After dialysis, centrifuge dialysis tube for 6 s at 500–1000 × g to collect sample (Fig 11D).
8.
Remove dialysis cap and replace with normal cap for storage.
The protein concentration of the sample is best determined using the 2-D Quant Kit. The kit allows accurate
quantitation of protein in the presence of detergents, reductants, and chaotropes that are incompatible with
other assays. See section 1.7.1 for a protocol describing the use of 2-D Quant Kit.
80-6429-60 AD 33
Dialysis solution
A substantial reduction in interfering ions can be achieved by dialyzing 2-D samples against a solution volume at least
40 times the sample volume, for 2 h to overnight.
Dialyze the sample against a solution that has the same concentrations of chaotropes (urea and thiourea) and DTT as
the sample. Other more expensive solution components such as CHAPS and carrier ampholytes do not need to be
included in the dialysis solution. These components may be added to their required concentrations following dialysis.
Samples for 2-D electrophoresis should be prepared in a solution that will be compatible with first-dimension IEF,
including urea, CHAPS, and DTT. See section 1.6.1 for details.
Vivaspin
Vivaspin concentrators can also be used for desalting samples
1.5.2 Removing undesirable nucleic acids from samples using Nuclease Mix
Removal of nucleic acids is often required to avoid contamination and subsequent artifacts on 2-D gels. Nuclease Mix
offers an effective cocktail of bovine pancreatic DNase and RNase enzymes, together with the necessary cofactors
for optimal nuclease activity. Nuclease Mix can be used together with Protease Inhibitor Mix since the latter does not
contain EDTA, an inhibitor of nuclease activity.
Protocol: Nuclease Mix
Components supplied
Nuclease Mix (100× solution), 0.5 ml. Each Nuclease Mix contains 4 µg of DNase (bovine pancreas) and 1 µg of RNase
(bovine pancreas) per µl solution.
Required but not provided
Vortex mixer.
1. Vortex briefly before taking an aliquot, as Nuclease Mix is supplied as a suspension.
2. Add 10 µl of Nuclease Mix per 1 ml reaction mix. Vortex briefly and incubate at room temperature for 30–45 min.
1.5.3 Simultaneous DNA, RNA and protein isolation from undivided
scarce samples
The illustra triplePrep Kit is designed for the rapid extraction of genomic DNA, total RNA and total denatured protein
from a single undivided sample. This kit can be used for removing undesirable nucleic acids. For more information on
protocol, see product booklet, 28-9425-44.
1.5.4 Using Albumin and IgG Removal Kit to improve 2-D electrophoresis
of human serum
Proteins in serum and other biological fluids are difficult to resolve by 2-D electrophoresis, largely due to the
abundance of serum albumin and IgG. In human serum, albumin constitutes 50–70% of the total protein and IgG
constitutes 10–25%. The presence of these proteins obscures other proteins in the gel and limits the amounts of
proteins in the serum that can be resolved by 2-D electrophoresis. In addition, these proteins have wide pI and
molecular weight ranges that further reduce resolution and mask some low-abundance proteins.
Albumin and IgG Removal Kit improves resolution of low-abundance proteins and increases the number of spots in
the treated sample. The kit includes an affinity gel that selectively binds human albumin and IgG and enhances the
visibility of low-abundance proteins. Albumin and IgG Removal Kit improves on the currently available Cibacron Blue
dye-based technology, which lacks selectivity and can remove low-abundance proteins of interest. See Figures 12
and 13 for typical results. Figure 14 depicts the methodology used in the kit.
34 80-6429-60 AD
Lane 1: Human Serum Albumin
Lane 2: Human IgG
Lane 3: Untreated serum
Lane 4: Treated serum replicate 1
Lane 5: Untreated serum
Lane 6: Treated serum replicate 2
Lane 7: Untreated serum
Lane 8: Treated serum replicate 3
Lane 9: Untreated serum
Lane 10:Treated serum replicate 4
Fig 12. Typical results when using the Albumin and IgG Removal Kit. Four replicates of a human serum sample were treated with
Albumin and IgG Removal Kit using the standard protocol (untreated samples in lanes 3, 5, 7, and 9, and treated samples in lanes 4, 6,
8, and 10). Untreated human serum was diluted to an equivalent volume. Equivalent amounts of untreated and treated serum were
separated onto a 12% acrylamide gel alongside purified albumin and IgG. The gel was stained with Sypro™ Ruby and imaged on
Typhoon™ 9400 Variable Mode Imager.
Untreated human serum
Human serum treated with Albumin and IgG Removal Kit
Fig 13. Removal of albumin from human serum. Following treatment with Albumin and IgG Removal Kit, albumin is removed and
lower-abundance proteins gain increased resolution. The albumin region of the gel before and after treatment is shown above.
CENTRIFUGE
Add
serum
Add
resin
Discard
upper
chamber
Incubate Transfer slurry
with
to upper
mixing
chamber of
MicroSpin column
Concentrate/desalt
sample as required
Fig. 14. Schematic of the removal process. Optimal albumin and IgG binding (>95% total protein) is achieved using a 15 μl human
serum loading and will typically lead to recovery of between 150 and 220 μg of lower-abundance proteins. The amount of protein
recovered will vary with the protein content of the individual serum samples used.
Protocol: Albumin and IgG Removal Kit
Components supplied
8.5 ml of a 50/50 (v/v) resin slurry, 10 empty microspin columns, 10 microcentrifuge tubes and lids.
Some of the components contain sodium azide in dilute solution. This substance is classified as toxic when
undiluted. Follow all local safety regulations when disposing of waste. Unless local regulations dictate other
methods, dispose of waste by flushing with copious amounts of water to avoid the build-up of explosive
metallic azides in copper and lead plumbing.
Required but not provided
Microcentrifuge, rotary shaker, disposable 15-ml centrifuge tube.
If performing acetone precipitation for concentration/desalting, acetone and acetone-resistant microcentrifuge tubes
are also required. Acetone precipitation is not necessary if using the 2-D Clean-Up Kit.
Preliminary notes
Ensure that the resin is in suspension when removing resin aliquots. Vigorous swirling of the resin bottle,
before the removal of each aliquot is recommended, to ensure the resin remains in suspension. Remove
an aliquot quickly using a wide-mouthed pipette tip.
80-6429-60 AD 35
During serum/resin incubation, ensure that the resin is kept in suspension by adequate mixing on a
rotary shaker.
Ensure that centrifugation is performed at the correct g force, and for the required time. This allows all
the liquid to be eluted from the resin following sample treatment. The resin should appear dry following
centrifugation.
Use of the 2-D Clean-Up Kit makes acetone precipitation unnecessary. However, if performing an acetone
precipitation, do not over-dry acetone-precipitated protein pellets.
Guidelines for serum sample loading with the Albumin IgG Removal Kit are listed in Table 12. The values are those
seen when a typical human serum sample is treated using the Albumin and IgG Removal Kit. Human serum samples
contain widely varying levels of albumin and IgG, and the information below should be used for guidance only.
Table 12. Typical levels for removal of albumin and IgG.
Human serum sample volume
Typical level of albumin removed
Typical level of IgG removed
15 µl
> 95%
> 90%
30 µl
> 80%
> 80%
45 µl
> 60–70%
> 70%
1. Pipette 15 µl of human serum into a sample tube with lid.
Tubes to be used for sample incubation should be of adequate size to allow good mixing of the resin/serum
sample volume, to ensure the resin remains in suspension during the incubation period. Disposable 15-ml
centrifuge tubes are recommended.
2. Add 750 µl of the suspended slurry to the tube containing the sample. It is essential that the gel slurry is
homogenous (uniform suspension) prior to pipetting.
When dispensing the resin it may be easier to pipette if the narrow end of the tip is cut off. This would
normally be performed with scissors prior to use.
3. Mix the gel/sample mixture on a rotary shaker for a minimum of 30 min at room temperature. Mixing speed
should be sufficient to keep the gel/sample in suspension.
Rapid rotary shaking is required to ensure that the resin remains in suspension. Speeds in the region of
250 rpm are recommended.
4. Snap off the base tip of the microspin column. Place each column into a microcentrifuge tube (supplied).
5. At the end of the incubation period, make sure that the resin is in suspension, and carefully pipette the gel/sample
mixture into the upper chamber of the microspin column, which is sitting in a microcentrifuge tube.
Ensure that all liquid is removed from the tube. If drops of liquid are splashed around the incubation tube,
briefly centrifuge the incubation tube and contents (1000 rpm, 2 min) to collect the resin and liquid, prior to
transfer to the microspin column.
6. Centrifuge at approximately 6500 × g for 5 min.
Note: The resin should appear dry and powdery following centrifugation.
7. Discard the upper chamber containing the gel, and collect the filtrate.
Note: The approximate volume of the filtrate will be 500 µl.
8. The sample is now ready to be used immediately for further processing or stored for later use.
If proteins are to be analyzed by 2-D gel analysis, concentration and desalting will be required.
Protocol for acetone precipitation (not required if using 2-D Clean-Up Kit)
1. Place an aliquot of acetone at -20 °C at least 15 min prior to use.
Approximately 2 ml of acetone is required for each sample. Ensure that the tube is made of an acetonecompatible material.
2. Accurately measure the volume of each sample. Divide each sample volume into two microcentrifuge tubes.
Add 4 volumes of ice cold acetone to the sample volume in each tube.
36 80-6429-60 AD
1.5-ml hinged microcentrifuge tubes are recommended. Small pellets are easily visualized using this
type of tube.
3. Allow proteins to precipitate at -20 °C for at least 2 h.
4. Pellet proteins by centrifugation at approximately 13 000 × g (13 000 rpm in a small bench microcentrifuge)
for 10 min at 2–8 °C.
Place the tubes into the microcentrifuge in known orientation. Place the hinged lid outward, as this assists
with detection of small protein pellets.
5. Decant off the acetone, but do not disturb the pellet. A clean tissue can be used to carefully remove any spots of
acetone away from the pellet.
6. Allow pellets to air dry (typically 5–10 min at room temperature). Do not over-dry.
Over-dried pellets can be difficult to resuspend.
7. Resuspend precipitated samples in an appropriate buffer as required.
Note: Typical levels of protein recovered from a 15 µl serum sample are 150–220 µg.
8. If samples are not to be used immediately, store at -20 °C or -70 °C until required.
1.6 Composition of sample preparation solution
In order to achieve a well-focused first-dimension separation, sample proteins must be completely disaggregated
and fully solubilized. Regardless of whether the sample is a relatively crude lysate or additional sample precipitation
steps have been employed, the sample solution must contain certain components to ensure complete solubilization
and denaturation prior to first-dimension IEF. These always include urea and one or more detergents. Complete
denaturation ensures that each protein is present in only one configuration and that aggregation and intermolecular interaction is avoided.
1.6.1 Components of sample preparation solutions
The role of each component of the sample solution is described below, as well as the recommended
concentration range.
Denaturant
IEF performed under denaturing conditions gives the highest resolution and the cleanest results. Urea, a neutral
chaotrope, is used as the denaturant in the first dimension of 2-D electrophoresis and is always included in the 2-D
sample solution at a concentration of at least 8 M. Urea solubilizes and unfolds most proteins to their fully random
conformation, with all ionizable groups exposed to solution. Recently, the use of thiourea in addition to urea has been
found to further improve solubilization, particularly of membrane proteins (10, 16, 55–57).
Detergent
A nonionic or zwitterionic detergent is always included in the sample solution to ensure complete sample solubilization and
to prevent aggregation through hydrophobic interactions. Originally, either of two similar nonionic detergents, NP-40
or Triton X-100, was used (1, 2). Subsequent studies have demonstrated that the zwitterionic detergent CHAPS (2–4%)
is often more effective (58) for solubilizing a wide range of samples. New zwitterionic detergents have been developed
and are reported to improve the solubility of membrane proteins (59, 60).
When difficulties in achieving full sample solubilization are encountered, the anionic detergent SDS can be used as
a solubilizing agent. SDS is a very effective protein solubilizer, but because it is charged and forms complexes with
proteins, it cannot be used as the sole detergent for solubilizing samples for 2-D electrophoresis. A widely used
method for negating the interfering effect of SDS is dilution of the sample with a solution containing an excess of
CHAPS, Triton X-100, or NP-40. The final concentration of SDS should be 0.25% or lower and the ratio of the excess
detergent to SDS should be at least 8:1 (27, 34, 61).
80-6429-60 AD 37
Reductant
Reducing agents are frequently included in the sample preparation solution to break any disulfide bonds present
and to maintain all proteins in their fully reduced state. The most commonly used reductant is dithiothreitol (DTT)
at concentrations ranging from 20 to 100 mM. Dithio-erythreitol (DTE) is similar to DTT and can also be used as a
reducing agent. Originally, 2-mercaptoethanol was used as a reductant, but higher concentrations of this reductant are
required and inherent impurities may result in artifacts (62). More recently, the non-thiol reductant tributyl phosphine
(TBP), at a concentration of 2 mM, has been used as a reductant for 2-D samples (63). However, due to the limited
solubility and instability of TBP in solution, a thiol reductant such as DTT must also be added to maintain proteins in
their reduced state through rehydration and first-dimension IEF. Reductants should be added directly before use.
Use of DeStreak Reagent is recommended for basic proteins. See section 2.6.2 for details.
Solubilizing agent
Carrier ampholytes or IPG Buffer (up to 2% [v/v]) can be included in the sample solution. They enhance protein
solubility by minimizing protein aggregation due to charge-charge interactions. In some cases, buffers or bases (e.g.
40 mM Tris) are added to the sample solution. This is done when basic conditions are required for full
solubilization or to minimize proteolysis. However, introduction of such ionic compounds can result in first-dimension
disturbances. Bases or buffers should be diluted to 5 mM or lower prior to loading the sample onto first-dimension IEF.
1.6.2 Examples of sample preparation solutions
A widely used sample solution, which can be used for initial experiments with an unknown sample, is described in
appendix I, solution A. To solubilize more hydrophobic proteins, use solution B in appendix I. For a general review of
protein solubilization for electrophoretic analysis, see reference 9.
1.7 Quantitating protein samples
Electrophoresis of protein samples requires accurate quantitation of the sample to be analyzed to ensure that
an appropriate amount of protein is loaded. In addition, accurate quantitation facilitates comparison between
similar samples by allowing identical amounts of protein to be loaded. Accurate quantitation of samples prepared
for electrophoresis is, however, difficult because many of the reagents used to prepare and solubilize samples for
electrophoresis, including detergents, reductants, chaotropes, and carrier ampholytes, are incompatible with common
protein assays.
Current spectrophotometric methods for protein quantitation rely either on Coomassie brilliant blue binding (65) or
protein-catalyzed reduction of cupric (Cu2+) ion to cuprous (Cu+) ion (66–68). Dye-binding assays cannot be used in the
presence of any reagent that also binds Coomassie brilliant blue. This includes carrier ampholytes such as Pharmalyte
and IPG Buffer and detergents such as CHAPS, SDS, or Triton X-100. Assays that depend on the reduction of cupric ions
cannot be used in the presence of reductants such as DTT, or in the presence of reagents that form complexes with
cupric ions such as thiourea or EDTA.
Samples prepared for IEF and SDS gel electrophoresis are often difficult to quantitate due to the presence of detergent
and reductant. Samples for 2-D electrophoresis are particularly difficult to quantitate due to the possible presence
of interfering carrier ampholyte and thiourea in addition to the detergents and reductants typically used in sample
preparation.
2-D Quant Kit (designed for the accurate determination of protein concentration in samples to be analyzed by highresolution electrophoresis) circumvents these limitations and can be used to accurately quantitate protein samples
prepared for 2-D electrophoresis. The procedure uses a combination of a unique precipitant and co-precipitant to
quantitatively precipitate sample protein while leaving interfering contaminants in solution. The protein is pelleted
by centrifugation and resuspended in an alkaline solution of cupric ions. The cupric ions bind to the polypeptide
backbones of any protein present. A colorimetric agent that reacts with unbound cupric ions is then added. The
color density is inversely related to the concentration of protein in the sample and the protein concentration can be
accurately estimated by comparison to a standard curve. Since the assay does not depend on reaction with protein
side-groups, reactivity is largely independent of amino acid composition. There is little protein-to-protein variation
using this assay.
38 80-6429-60 AD
1.7.1 Protein determination using 2-D Quant Kit
2-D Quant Kit is designed to accurately determine protein concentrations in samples for electrophoresis. Proteins are
quantitatively precipitated while interfering substances are left in solution. The color density that develops in the 2-D
Quant Kit procedure is inversely related to the protein concentration, with a linear response to protein in the range of
0–50 µg and a volume range of 1–50 µl. The procedure is compatible with common sample preparation reagents listed
in Table 13.
Table 13. Compounds tested for assay compatibility.
Compound
Concentration
SDS 2% (w/v)
CHAPS 4% (w/v)
Triton X-100 1% (w/v)
Pharmalyte pH 3–10 2% (v/v)
IPG Buffer pH 3–10 NL 2% (v/v)
Tris 50 mM
EDTA 10 mM
DTT 1% (65 mM)
2-mercaptoethanol 2% (v/v)
Urea
8M
Thiourea 2M
Glycerol 30% (w/v)
Standard curve with BSA dissolved in water
Standard curve with BSA dissolved in first-dimension
sample solution (8 M urea, 4% CHAPS, 40 mM DTT,
2% Pharmalyte pH 3–10)
0.8
A 480
0.7
0.6
0.5
0.4
0
10
20
30
µg BSA
40
50
Fig 15. The 2-D Quant Kit protein assay eliminates interference from sample solution components.
Protocol: 2-D Quant Kit
Components supplied
Precipitant, co-precipitant, copper solution, color reagent A, color reagent B, bovine serum albumin (BSA)
standard solution.
Required but not provided
Microcentrifuge, microcentrifuge tubes (2 ml), vortex mixer, visible-light spectrophotometer, spectrophotometer cells.
Preliminary notes
Prior to starting procedure, prepare working color reagent by mixing 100 parts of color reagent A with one part of
color reagent B. Each individual assay requires 1 ml working color reagent.
1. Prepare standard curve (0–50 µg) using the 2 mg/ml BSA standard solution.
2. Prepare microcentrifuge tubes (in duplicate) containing 1–50 µl of the sample to be assayed.
The useful range of the assay is 0.5–50 µg.
80-6429-60 AD 39
3. Add 500 µl precipitant to each microcentrifuge tube (including tubes containing the BSA standard).
Vortex and incubate 2–3 min at room temperature.
4. Add 500 µl co-precipitant. Mix briefly.
5. Centrifuge (at least 10 000 × g) for 5 min.
6. Remove supernatant. Centrifuge briefly to bring remaining supernatant to bottom of tube. Remove remaining
supernatant with micropipette.
Proceed rapidly to avoid resuspension or dispersion of pellet. There should be no visible liquid remaining.
7. Add 100 µl copper solution and 400 µl distilled or deionized water to each tube. Vortex to dissolve the
precipitated protein.
Ensure that the pellet is completely resuspended by vortexing thoroughly.
8. Add 1 ml working color reagent to each tube. Ensure instantaneous mixing by introducing the reagent as rapidly
as possible.
9. Incubate at room temperature for 15–20 min.
10.Read the absorbance at 480 nm for each sample and standard using a spectrophotometer such as
Ultrospec1100 pro UV/Visible Spectrophotometer.
11.Generate standard curve by plotting the absorbance of the standards against the quantity of protein.
12.Estimate protein concentration of samples by comparison to the standard curve.
1.8 Sample loads
The optimal quantity of protein to load for electrophoresis varies widely depending on factors such as sample
complexity, the length and pH range of the Immobiline DryStrip gel, and the method of visualizing the 2-D separation.
General sample load guidelines for different staining techniques are given in chapter 2, Table 16.
40 80-6429-60 AD
2. First-dimension isoelectric focusing (IEF)
2.0 Overview
GE Healthcare offers two flatbed electrophoresis systems for first-dimension separation using isoelectric focusing
(IEF): Ettan IPGphor 3 Isoelectric Focusing System and Multiphor II Electrophoresis System. This chapter provides
information on Ettan IPGphor 3; information specific to Multiphor II is covered in chapter 4.
Ettan IPGphor 3 Isoelectric Focusing System comprises Immobiline DryStrip gel strips, which contain an
immobilized pH gradient (IPG) and are commonly referred to as IPG strips; two accessory options for holding the
strips in place— the Manifold and fixed-length Strip Holders; and the Ettan IPGphor 3 unit, which includes a highvoltage DC power supply, solid state temperature control using Peltier technology, and programming options for up
to 10 user-defined IEF protocols.
IPG strips are available in five lengths—7, 11, 13, 18, and 24 cm—and a number of pH ranges, both linear and
nonlinear. Section 2.2 discusses choices for length of strip, pH gradient, and buffer.
The Manifold accommodates IPG strips from 7 to 24 cm long, and holds up to 12 strips. It allows for three main
means of sample application:
(1) rehydration loading, generally for preparative or analytical loadings of broad-range or narrow-range strips;
(2) cup loading (anodic or cathodic), generally for analytical loadings of basic strips or very acidic strips,
respectively; and
(3) paper-bridge loading (anodic or cathodic), generally for preparative loadings of basic strips or very acidic strips,
respectively.
Further discussion of these techniques can be found in section 2.7.
The Manifold must be used in conjunction with the DryStrip IPGbox (with or without sample included) in order to
rehydrate the Immobiline DryStrips (see section 2.7). The Manifold can also be used for equilibrating the IPG strips
prior to second-dimension electrophoresis.
The regular Strip Holder (see section 2.7) is placed on the Ettan IPGphor 3 electrode platform, and the sample
is introduced either during or after the rehydration step. Up to 12 Strip Holders of the same length can be
accommodated for any one protocol.
An earlier product, the Cup Loading Strip Holder, is not included in the discussion that follows.
2.1 Background to isoelectric focusing
Isoelectric Focusing is an electrophoretic method that separates proteins according to their isoelectric points (pI).
Proteins are amphoteric molecules; they carry either positive, negative, or zero net charge, depending on the pH
of their surroundings (Fig 16). The net charge of a protein is the sum of all the negative and positive charges of its
amino acid side chains and amino- and carboxyl-termini. The isoelectric point (pI) is the specific pH at which the net
charge of the protein is zero. Proteins are positively charged at pH values below their pI and negatively charged at
pH values above their pI. If the net charge of a protein is plotted versus the pH of its environment, the resulting curve
intersects the x-axis at the isoelectric point (Fig 16).
COO
COOH
+
COO
+
NH3
COO
COO
NH2
NH3
pH=pI
+2
Isoelectric point (pl)
+1
-
+
+
NH3
Net Charge
+3
-
NH2
NH3
COOH
pH<pI
-
pH>pI
-
0
3
4
5
6
7
8
9
10
11 pH
-1
-2
-3
Fig 16. Plot of the net charge of a protein versus the pH of its environment. The point of intersection of the curve at the x-axis
represents the isoelectric point of the protein.
80-6429-60 AD 41
The presence of a pH gradient is critical to the IEF technique. In a pH gradient and under the influence of an electric
field, a protein will move to the position in the gradient where its net charge is zero. A protein with a net positive
charge will migrate toward the cathode, becoming progressively less positively charged as it moves through the pH
gradient until it reaches its pI. A protein with a net negative charge will migrate toward the anode, becoming less
negatively charged until it also reaches zero net charge. If a protein should diffuse away from its pI, it immediately
gains charge and migrates back. This is the focusing effect of IEF, which concentrates proteins at their pIs and allows
proteins to be separated on the basis of very small charge differences.
The resolution is determined by the slope of the pH gradient and the electric field strength. IEF is therefore performed
at high voltages (typically in excess of 1000 V). When the proteins have reached their final positions in the pH gradient,
there is very little ionic movement in the system, resulting in a very low final current (typically in the microamp range).
IEF of a given sample in a given electrophoresis system is generally performed for a constant number of Volt-hours
(Volt-hour [Vh] being the integral of the volts applied over the separation time).
IEF performed under denaturing conditions gives the highest resolution and the sharpest results. Complete
denaturation and solubilization is achieved with a mixture of urea, detergent, and reductant, ensuring that each
protein is present in only one conformation with no aggregation, therefore minimizing intermolecular interactions. See
section 1.6 for a discussion of the components of sample preparation solutions.
The original method for first-dimension IEF depended on carrier-ampholyte-generated pH gradients in cylindrical
polyacrylamide gels cast in glass rods or tubes (1). Carrier ampholytes are small, soluble, amphoteric molecules with
a high buffering capacity near their pI. When a voltage is applied across a carrier ampholyte mixture, the carrier
ampholytes with the highest pI (and the most negative charge) move toward the anode; those with the lowest pI (and
the most positive charge) move toward the cathode. The other carrier ampholytes align themselves between the
extremes according to their pIs, and buffer their environment to the corresponding pHs. The result is
a continuous pH gradient.
As a result of limitations and problems associated with carrier ampholyte pH gradients, immobilized pH gradients
(IPG) were developed. GE Healthcare subsequently introduced Immobiline chemicals for the generation of this type
of pH gradient (2). Görg et al. (3, 4) pioneered the development and use of IPG IEF for the first-dimension of 2-D
electrophoresis.
An immobilized pH gradient is created by covalently incorporating a gradient of acidic and basic buffering groups
(immobilines) into a polyacrylamide gel at the time it is cast. Immobiline buffers are a set of well-characterized
molecules, each with a single acidic or basic buffering group linked to an acrylamide monomer.
The general structure of Immobiline reagents is:
CH2 CH–C–NH–R
O
R = weakly acidic or basic buffering group
Immobilized pH gradients are formed using two solutions, one containing a relatively acidic mixture of acrylamido
buffers and the other containing a relatively basic buffer mixture. The concentrations of the various buffers in the
two solutions define the range and shape of the pH gradient produced. Both solutions contain acrylamide monomers
and catalysts. During polymerization, the acrylamide portion of the buffers copolymerizes with the acrylamide and
bisacrylamide monomers to form a polyacrylamide gel. Figure 17 is a graphic representation of the polyacrylamide
matrix with attached buffering groups.
42 80-6429-60 AD
+
N
H
R
R
NH+
R
C
O–
O
R
C
O
–
O
N H+
R
R
Fig 17. Immobilized pH gradient polyacrylamide gel matrix showing attached buffering groups.
For improved performance and simplified handling, the Immobiline DryStrip gel is cast onto a plastic backing
(GelBond™ PAGfilm). The gel is then washed to remove catalysts and unpolymerized monomers that could otherwise
modify proteins and interfere with separation. Finally, the gel is dried and cut into 3-mm-wide strips. The resulting
Immobiline DryStrip gels can be rehydrated with a rehydration solution containing the necessary components for
first-dimension IEF.
IEF is performed with the Immobiline DryStrip gels using a flatbed electrophoresis unit such as Ettan IPGphor 3.
The advantages of using the flatbed format are:
• Since the pI of a protein is dependent on the temperature, precise cooling is required during IEF. This can be
effectively achieved by using the aluminum oxide ceramic Strip Holder or Manifold in conjunction with a Peltier
temperature-controlled bed.
• Since IEF requires high field strengths to obtain sharply focused bands, high voltages must be applied. A flatbed
design is the most economical way to meet the necessary safety standards required to operate at such high
voltages.
2.2 Immobiline DryStrip gels
Immobiline DryStrip gels offer a marked improvement over tube gels using carrier ampholyte–generated pH
gradients. When Immobiline DryStrip gels are used for the first-dimension separation, the resultant 2-D spot maps
provide superior results in terms of resolution and reproducibility:
• The first-dimension separation is more reproducible because the covalently fixed gradient cannot drift.
• Plastic-backed Immobiline DryStrip gels are easy to handle. They can be picked up at either end with forceps or
gloved fingers.
• The plastic support film prevents the gels from stretching or breaking.
• IPG technology increases the useful pH range on any single Immobiline DryStrip gel; more very acidic and basic
proteins can be separated.
• Immobiline DryStrip gels have a higher protein loading capacity (69).
• The sample can be introduced into the Immobiline DryStrip gel during rehydration (70, 71).
• Precast Immobiline DryStrip gels eliminate the need to handle toxic acrylamide monomers. In addition, preparation
time and effort are significantly reduced, and reproducibility of the pH gradient is ensured.
Additional advantages of Immobiline DryStrip gels include:
• Immobilized pH gradients and precise lengths ensure high reproducibility and reliable gel-to-gel comparisons.
• To simplify gel use and record keeping, each strip is labeled with the pH interval and a unique identifier, and
bar-coded for use with a reader. A “+” or “-” sign indicates the anodic or cathodic side of the strip, respectively.
80-6429-60 AD 43
• Immobiline DryStrip gels are compatible with Ettan DIGE system, the most powerful approach for comparative
analysis of relative protein abundance using 2-D electrophoresis (see chapter 6).
Figure 18 illustrates the pH intervals of Immobiline DryStrip gels. The most recent additions to the product line
comprise two sets of pH interval: a broad-range gel (pH 3–11 NL) and four medium-range gels covering shorter pH
intervals—pH 3–5.6 NL, 5.3–6.5, 6.2–7.5, and 7–11 NL. These four medium-range gradients overlap each other in an
optimized way, allowing outstanding coverage of all proteins in the pH 3 to 11 range, with improved separation in
the extreme basic pH region of the IPG strips. Narrow-range Immobiline DryStrip gels covering just one pH unit are a
valuable complement to the newer strips in many experimental situations.
Strip length
IPG Strips
24 cm
18 cm
13 cm
11 cm
7 cm
×
×
×
×
×
×
×
×
×
×
×
×
×
×
×
×
×
×
×
×
×
×
×
×
×
×
×
×
×
×
×
×
×
×
×
×
×
×
×
×
×
×
×
Narrow
3.5-4.5
5.3-6.5
6.2-7.5
Medium
3-5.6 NL
3-7 NL
4-7
6-9
6-11
7-11 NL
Wide
3-10
3-11 NL
3-10 NL
×
×
×
×
Fig 18. A comprehensive range of overlapping IPG strips covering narrow, medium, and wide pH ranges are available in several
different strip lengths.
DryStrip gels are rehydrated in a solution containing the necessary additives and, optionally, the sample proteins
(rehydration solution is described in detail in section 2.6). IEF is performed at high voltage. After IEF, the Immobiline
DryStrip gels are equilibrated in equilibration solution and applied onto vertical or flatbed SDS-polyacrylamide gels for
the second-dimension separation (see chapter 3).
After IEF, proceed to the second-dimension separation immediately or store the Immobiline DryStrip gels at
-60°C or below, as described in section 2.8.3.
2.2.1 Choosing strip length
Immobiline DryStrip gels are available with strip lengths of 7, 11, 13, 18, and 24 cm, with a precise gel length tolerance
of ±2 mm.
Choose shorter strips, i.e. up to 13 cm, for fast, cost-effective screening or when the most abundant proteins are of
highest interest (as in prefractionated protein complexes). The shortest IPG strips give the fastest results, but their
sample load is limited.
Use longer strips, i.e. 18- and 24-cm strips, for maximal resolution and loading capacity. Longer strips allow detection
of more spots and make it easier to select and identify the proteins in the map, but require longer times in both the
first- and the second-dimension separations. Table 14 shows the inter-relationship between these parameters. For the
highest possible resolution, use 24-cm strip lengths.
44 80-6429-60 AD
Table 14. Typical operating parameters for Immobiline DryStrip pH 4–7 gels with E. coli extract and analytical load. The number of
detectable spots is increased by roughly the same factor as the increase in separation length. The same relationship is true for other
pH intervals as well.
Parameters
7 cm
11 cm
13 cm
18 cm
2
4
4
6
10
Time second dimension (h)
1.5
2.5
3
5.5
5.5
Sample load (µg proteins) —analytical gels
10
25
30
55
90
Time first dimension (h)
24 cm
2.2.2 Choosing the pH gradient
Immobiline DryStrip gels allow effective IEF over a wide pH range, from very acidic proteins at pH 3 to extremely
basic proteins at pH 11. These varied pH intervals allow fine-tuning of each separation strategy to increase firstdimension loading and resolve a greater number of spots from crowded areas. Both aspects will improve later protein
identification and characterization.
To overview total protein distribution, use pH 3–11 NL strips (NL refers to nonlinear). The broad-range
pH 3–11 NL Immobiline DryStrip gel works with most protein mixtures from prokaryotic and eukaryotic cells.
Results obtained can be used as a basis for developing a more specific separation strategy using mediumrange pH gradients.
For increased resolution between pH 5 and pH 7, use a nonlinear gradient pH 3–10 strip (pH 3–10 NL)
to distribute the proteins more evenly over the strip. This is especially helpful when analyzing complex samples
like serum, cerebrospinal fluid, extracts from E. coli, and yeasts.
Combine pH 3–7 and pH 6–11, or pH 3–7 and pH 6–9, or select from pH 3–5.6 NL, 5.3–6.5, 6.2–7.5, or
7–11 NL when more detail is required. Of these, the two NL strips—pH 3–5.6 NL and 7–11 NL—are nonlinear
at the extreme ends of the pH scale, allowing a more even distribution of proteins over the gel length and
maximized resolution. The pH 3–5.6 NL, 5.3–6.5, 6.2–7.5, and 7–11 NL strips provide optimal overlaps, and
approximately the same number of proteins is separated in each pH interval. Higher sample loading capacity
of medium-range gels makes protein identification easier.
Use narrow-pH-range strips (1 pH unit) to closely study proteins in the regions of interest. Narrow gradients of
1 pH unit allow higher resolution, in-depth study of proteins separating in these regions, and increased loading
capacity. Several milligrams of protein extract can be analyzed when using rehydration loading (including
protein samples in the rehydration solution), which simplifies identification and characterization of spots in the
2-D map.
Note: To increase the stability of the pH gradient in Immobiline DryStrip pH 7–11 NL, during production the buffering
capacity is enhanced at the most basic end by the introduction of a proprietary arginine derivative (guanidyl group).
To avoid redox-related streaking, all the basic strips should be rehydrated with DeStreak Rehydration Solution
(see sections 2.6.1 and 2.6.2).
Note: The gradients overlap to enable the assembly of virtual high-resolution 2-D maps from different narrow-range
separations.
If a specialized pH gradient is required, recipes for preparing custom narrow- and wide-range immobilized pH
gradients are given in references 72 and 73.
Figure 19 shows typical results using broad- and medium-range, 24-cm Immobiline DryStrip gels.
80-6429-60 AD 45
Fig 19a. Broad-range Immobiline DryStrip pH 3–11 NL, 24 cm
using 0.5% IPG Buffer 3–11 NL, run for 42 kVh.
Sample: 100 µg mouse liver extract + 7.5 µg alkylated lysozyme,
cup application at the anode.
Fig 19b. Medium-range
Immobiline DryStrip
pH 3–5.6 NL, 24 cm using
2% IPG Buffer 3.5–5.0, run
for 50 kVh.
Sample: 140 µg mouse liver
extract, cup application at
the cathode.
Fig 19c. Medium-range
Immobiline DryStrip
pH 5.3–6.5, 24 cm using
2% IPG Buffer 5.5–6.7, run
for 116 kVh.
Sample: 100 µg mouse liver
extract, cup application at
the anode.
Fig 19d. Medium-range
Immobiline DryStrip
pH 6.2–7.5, 24 cm using
2% IPG Buffer 6–11, run
for 116 kVh.
Sample: 100 µg mouse liver
extract, cup application at
the anode.
Fig 19e. Medium-range
Immobiline DryStrip
pH 7–11 NL, 24 cm using
0.5% IPG Buffer 7–11 NL,
run for 75 kVh.
Sample: 100 µg of mouse
liver extract + 5 µg alkylated
lysozyme, cup application at
the anode.
Fig 19. Two-dimensional electrophoresis of mouse liver extract, with or without alkylated lysozyme, using broad- and medium range,
24-cm Immobiline DryStrip gels. All first-dimension results shown in Figure 19 were run on Ettan IPGphor 3 with Ettan IPGphor 3 Manifold.
Immobiline DryStrip gels were rehydrated in DeStreak Rehydration Solution with IPG Buffer solutions as indicated. Second dimension:
Ettan DALT twelve using DALT Gel 12.5 precast SDS-PAGE gels. Staining: PlusOne Silver Staining Kit, Protein. White rings = same protein
seen on two pH intervals i.e. overlaps between pH gradients. Rings or ovals of the same color present on the broad pH gradient 3–11 NL
(Fig 19a) and the medium-range gradients (Figs 19b–19e) indicate the same protein groups. Yellow ring = alkylated lysozyme, pI = 10.5
(calculated according to SwissProt).
2.2.3 Choosing an IPG Buffer
IPG Buffers are ampholyte-containing buffer concentrates specifically formulated for use with Immobiline DryStrip gels.
Each IPG Buffer type produces more uniform conductivity along the Immobiline DryStrip during focusing, resulting in wider
latitude in run times. IPG Buffers also eliminate potential high background staining. The buffers, supplied in 1-ml aliquots,
are diluted 50- or 200-fold in the rehydration solution, depending on the first-dimension system and pH range of the
strip. Figure 20 shows the appropriate IPG Buffer for use with the various IPG DryStrip gels.
2.2.4 Estimating the pI of proteins
The reliability of the first-dimension separation is so high that the pI of a protein can be estimated by relating its
position on the second-dimension gel to its original position on the Immobiline DryStrip. Using linear pH gradients
increases the accuracy of this estimation.
Determine the first-dimension position by measuring the length of the Immobiline DryStrip gel and the position of the
spot on the second-dimension gel (for gels not attached to backing, correct for shrinkage or swelling of the gel during
staining). Then plot the spot position (as a percent of gel length) versus pH and read off the pI from the graph of the pH
gradient found in Data File 18-1177-60 (see additional reading and reference material).
46 80-6429-60 AD
pH range
IPG Buffers
Narrow
3.5-4.5
5.3-6.5
6.2-7.5
Medium
3-5.6 NL
3-7 NL
4-7
6-9
6-11
7-11 NL
Wide
3-10
3-11 NL
3-10 NL
3.5-5.0 5.5-6.7
4-7
6-11
7-11 NL 3-10 NL
3-10
3-11 NL
Fig 20. Using IPG strips together with matching IPG Buffer improves the conductivity distribution across the pH gradient during IEF.
2.3 IEF using Ettan IPGphor 3 Isoelectric Focusing
System and accessories
Ettan IPGphor 3 (Fig 21) is a fully integrated IEF system optimized to deliver high throughput, speed, reproducibility,
and high protein-loading capacity, as well as optional PC control. The large graphical display accommodates multiple
(up to four) lines of text for fast and easy programming. Up to10 protocols (nine steps each) can be saved, retrieved,
and easily edited on the instrument. Any number of protocols can be stored on a connected PC running IPGphor 3
Control Software and uploaded to the instrument instantly. Important safety features ensure safe high-voltage runs.
Key accessories include Strip Holders, IPGbox, and Manifold. Ettan IPGphor 3 provides:
• Integral 10 000 V power supply
• Peltier solid-state temperature control (18–25 °C)
• Accommodation of one Manifold or 1–12 Strip Holders for 7-, 11-, 13-, 18-, or 24-cm IPG strips
• Programmable controller for voltage, current, temperature, and time
Fig 21. Ettan IPGphor 3.
80-6429-60 AD 47
2.3.1 Ettan IPGphor 3 Control Software
Ettan IPGphor 3 Control Software (Fig 22), with an external personal computer (Windows) connected via a serial port,
can be used to control up to four Ettan IPGphor 3 units simultaneously, each running a different set of run parameters.
With the software it is possible to:
• Create, save, and edit protocols
• Monitor voltage, current, and volt-hours of the run and generate graphical display as the run proceeds
• Open and view stored log files of previous runs
• Start, stop, and pause Ettan IPGphor 3
• Generate status report on Ettan IPGphor 3 (instantaneous run condition report on request)
• Enable Web browser remote monitoring of IPGphor 3
• Export log files to programs such as Microsoft™ Excel™
• Create professional reports that can be saved, printed, and exported
Fig 22. Ettan IPGphor 3 Control Software. For detailed instructions on installation and usage of this software, refer to the
Ettan IPGphor 3 Control Software user manual.
2.3.2 Ettan IPGphor 3 Manifold
Ettan IPGphor 3 Manifold is an accessory for first-dimension IEF of proteins on IPG strips. The Manifold is designed to
handle IEF and subsequent equilibration for up to 12 IPG strips. All strips in a given run must be of the same length. The
Manifold can accommodate all IPG strip sizes from 7 to 24 cm. It comes with a complete set of accessories for 10 full
runs of 12 strips each. It is also compatible with the first-generation Ettan IPGphor (see Ettan IPGphor user manual).
Cup-based sample application can improve protein-focusing patterns, particularly in basic IPG strips, and the
Manifold accommodates either anodic or cathodic loading. Each cup can hold sample volumes of up to 150 µl.
Under conditions where substantial water transport (electroendosmosis) accompanies focusing, such as with basic
strips or with protein loads in excess of 1 mg, the face-up mode frequently yields improved resolution. Running strips
gel side up has a number of advantages over the use of regular Strip Holders:
48 80-6429-60 AD
• It is easy to apply filter wicks at the electrodes: With preparative loads there is a more pronounced movement
of water (due to electroendosmotic effects resulting from the extra proteins and potentially more salt/buffer
carryover). This water movement is also more pronounced when working with basic IPG strips (pH ranges 6–9,
6–11, and 7–11 NL). Moistened prior to use, the paper wicks have the ability to absorb excess ions and buffers
that move to the electrodes and that may otherwise perturb the focusing. They also serve to absorb the water
accumulating at the cathodic side of the strip (as H3O+) and to keep the anodic side of basic strips hydrated
(potentially they can dry out from the depletion of water). Moreover, since preparative loads are usually applied to
narrow-range strips, the wicks will soak up the excess proteins that lie above and below the pH range
being studied.
• It is easy to apply a cup to the surface of the gel for sample application: Cup loading can be advantageous for basic
proteins (and also for very acidic proteins), mostly due to stability issues with these proteins once they reach their pI.
Sharper spots can be obtained by loading samples away from their pI. Thus basic proteins would be loaded at the
anodic side and acidic proteins at the cathodic side.
• The electrodes are fully adjustable to suit the length of strip: The Manifold can be used to run any IPG strip with
a length between 7 and 24 cm. One to 12 strips, all of the same length, can be run at the same time.
• No pressure is exerted against the strip surface: Because the gel is run face side up, there is no pressure of the gel
against the ceramic surface (such as in the regular Strip Holder). This is advantageous when running preparative
loads as it lessens the streaking associated with abundant proteins.
There are, however, several drawbacks to running strips gel side up: the inability to apply voltage during rehydration;
the extra manipulation of the strip from tray to Manifold; and the absence of active temperature control during the
rehydration step.
For best results with the basic IPG strips, in addition to anodic cup-loading, the use of DeStreak Reagent is
highly recommended (see section 2.6.2).
The Manifold tray base is made of a thermally conductive aluminum oxide ceramic that rapidly dissipates heat to
avoid potential “hot spots.” A further special coating of the surface eliminates protein absorption. The Manifold tray
allows simple and accurate placement of IPG strips, with protrusions along the numbered inner channels that keep
IPG strips straight and centered.
Pre-cut electrode pads and paper bridges are convenient and save valuable time. The wicks absorb excess water,
salts, and proteins while the paper bridges can be used to load large sample volumes.
The overall procedure for use of Ettan IPGphor 3 with the Manifold is depicted in Figure 23. Following sections provide
protocols for use of the various accessories.
After IEF, proceed to the second-dimension separation immediately or store the Immobiline DryStrip gels at
-60 °C or below, as described in section 2.8.3.
The Ettan IPGphor 3 platform is available in three application-based options for high-throughput analytical and
micropreparative protein analysis. Table 15 summarizes these options.
Table 15. Several options based on application.
The complete solution
Ettan IPGphor 3 Isoelectric Focusing Unit plus Ettan IPGphor Manifold,
IPGbox, ceramic Strip Holders to run 7-, 11-, 13-, 18-, or 24-cm
IPG strips, and appropriate IPG strips and buffers
For high-throughput micropreparative applications
Ettan IPGphor 3 Isoelectric Focusing Unit plus Ettan IPGphor Manifold,
IPGbox, and appropriate IPG strips and buffers
For analytical study of protein profiles
Ettan IPGphor 3 Isoelectric Focusing Unit plus ceramic Strip Holders
and appropriate IPG strips and buffers
80-6429-60 AD 49
1. Rehydrate IPG strips
Rehydrate Immobiline DryStrip
gels, with the gel side down,
in the appropriate volume of
rehydration solution, using
IPGbox. Allow the IPG strips to
rehydrate overnight (10–20 h).
2. Position Manifold
Position the Manifold on Ettan
IPGphor 3. The small T-shaped
protrusion fits into the cutout
section of the Ettan IPGphor 3
bed, making positioning easy.
3. Transfer IPG strips to
Manifold
Pour the appropriate volume
(108 ml) of Immobiline DryStrip
Cover Fluid evenly in all the
channels. Transfer the IPG
strips to the Ettan IPGphor 3
Manifold. Place them face up
in the tray with the anodic (+)
end of the strip resting on the
appropriate mark etched on the
bottom of the Manifold track.
Wet the precut electrode pads
with 150 µl deionized water
and place the pads on the
ends of the IPG Strips.
6. Position electrode
assembly
Slide an electrode assembly
over the top of all the pads.
Swivel the cams into the
position under the external
lip of the Manifold to seat the
electrode in place.
7. Load and cover samples
Load the samples into
the sample cups, up to a
maximum of 150 µl. Check to
make sure that the samples
are completely covered with
DryStrip Cover Fluid.
4. Seat cups in track (if cup
loading)
8. Set program parameters
and run
Place a strip of cups in the
appropriate position. The
convenient seating tool
enables you to push the cups
down so that they are properly
seated at the bottom of the
track.
Close the Ettan IPGphor 3
cover. Select program and
enter desired run parameters
and begin the run.
Fig 23. Summary of the steps involved in first-dimension IEF using Ettan IPGphor 3, IPGbox, and Manifold.
50 80-6429-60 AD
5. Moisten and place
electrode pads
2.3.3 IPGbox
IPGbox and IPGbox Kit are tools for enhancing the reswelling of GE Healthcare’s precast Immobiline DryStrip Gels.
It provides a convenient method for rehydrating up to twelve precast IPG strips (7 to 24 cm) at a time. Individual
slots in the Reswell Trays allow rehydration of individual IPG strips in a minimum volume of solution. The IPGbox
includes a lid that protects the strips from dust and other contaminants during the rehydration period, which
ranges from 10 h to overnight.
2.3.4 Ettan IPGphor 3 Strip Holders
IPGphor Strip Holder serves as both a rehydration and focusing chamber for individual IPG strips. When the sample is
included in the rehydration solution, it is loaded into the gel by absorption during the rehydration step. Since the gel
is in direct contact with electrodes built into the Strip Holder, it is placed in position to run without further handling. The
base of the Strip Holder is made from the same thermally conductive aluminum oxide ceramic as the Manifold and
has platinum electrodes at each end. The transparent Strip Holder cover allows easy visual monitoring of rehydration
and focusing progress.
2.3.5 General cautions
Ettan IPGphor 3 is a high-voltage instrument that can cause fatal electrical shock if the safety features are
disabled. As such, the safety lid must be properly latched before starting a protocol, otherwise voltage will not
be applied.
Exceeding the recommended current limit of 75 µA per IPG strip can cause the strip to burn and may damage
the instrument.
During isoelectric focusing, do not lean on the safety lid, do not apply excess pressure or uneven weight to
the lid, and do not place any items on the lid. Such pressure could cause arcing between the Strip Holder electrodes and the electrode areas, damaging the instrument.
The Strip Holders and Manifold trays are made of ceramic and should be handled carefully.
Always wear gloves when handling IPG strips and the equipment that comes in contact with them. This will
help minimize protein contamination, which can result in artifactual spots in the resulting 2-D spot patterns.
Clean Strip Holders and Manifold with the Strip Holder cleaning solution provided or the protective coating
will be compromised. Clean all other components that come in contact with the IPG strip or the sample with a
detergent designed for glassware. Rinse well with distilled water.
Use the appropriate rehydration volume for the IPG strip length (refer to appropriate protocol).
Do not heat any solutions containing urea above 30 ºC as isocyanate, a urea degradation product, will
carbamylate the proteins in the sample, thus changing their isoelectric points.
All chemicals should be of the highest purity (electrophoresis grade or better), and water should be double
distilled or deionized.
80-6429-60 AD 51
2.4 Selecting sample application method
Sample can be applied either by including it in the rehydration solution (rehydration loading) or by applying it directly
to the rehydrated Immobiline DryStrip gel via sample cups or a paper bridge.
2.4.1 Rehydration loading
Rehydration loading (see section 2.7) offers such advantages as loading and separation of larger sample volumes
(greater than 100 µl) (70, 71), larger sample amounts, and more dilute samples. Because there is no discrete
application point, this method eliminates the formation of precipitates at the application point that may occur when
loading using sample cups. Also, the method is technically simpler than the others, avoiding problems of leakage that
can occur when using sample cups.
2.4.2 Use of Manifold
There are cases when it may be preferable to load the sample following rehydration, immediately prior to IEF. For
example, if proteolysis or other protein modifications are a concern, overnight rehydration with sample may not be
desirable. The Manifold (see section 2.7) provides a convenient means to load samples under such circumstances. Cup
loading using the Manifold is recommended for sample volumes up to 150 µl, and a maximum protein concentration
of 150 µg protein/150 µl sample solution (150 µl is the volume of the cup). Larger sample loads can lead to increased
protein precipitation at the point of application.
Anodic cup loading has been found to improve protein 2-D spot patterns with basic Immobiline DryStrip gels (pH 6–9,
pH 6–11, and pH 7–11 NL). Under conditions where substantial water transport (electroendosmosis) accompanies
focusing, such as with protein loads in excess of 1 mg, the face-up mode frequently yields better resolution. See
section 2.3.4 for a more detailed discussion of the face-up mode.
2.4.3 Paper-bridge loading
Paper-bridge loading is ideal for very large sample volumes and preparative electrophoresis, and is particularly
applicable when using basic pH intervals (pH 6–9, pH 6–11, and pH 7–11 NL).
Paper-bridge loading can also be performed in the Manifold. Using 18- or 24-cm Immobiline DryStrip gels, up to 450 µl
can be applied using the paper-bridge method.
Details of appropriate sample loads for analytical and preparative loading and cup loading using the Manifold are
given in Table 16 (see section 2.5).
Figure 24 gives general guidelines on selecting the appropriate mode of sample application.
Analytical
pH gradient
3.5–4.5
3.0-5.6 NL
4.0-7.0
3.0-7.0 NL
5.3-6.5
3.0-10.0
3.0-10.0 NL
3.0-11.0 NL
6.2-7.5
Strip Holder
Rehydration
loading
Preparative
Manifold
Rehydration
loading
Manifold
Cup
loading
6.0-9.0
6.0-11.0
7.0-11.0 NL
Rehydration paper-bridge
loading
loading
Cup
loading
paper-bridge
loading
Fig 24. Guidelines for selecting the appropriate mode of sample application in the Ettan IPGphor 3 Isoelectric Focusing System.
Refer to section 2.7 for more details on sample application.
52 80-6429-60 AD
2.5 Recommended sample loads
Recommended sample loads for silver (for analytical analysis) and Coomassie (for preparative analysis) staining are
shown in Table 16. 2-D Quant Kit (see section 1.7.1) can be used to determine the protein concentration prior to firstdimension IEF.
Table 16. Suitable sample loads* for silver and Coomassie staining using cup loading and rehydration loading.
Immobiline DryStrip gel Suitable sample load (µg of protein)
length (cm)
(pH)
Silver stain
Coomassie stain
(analytical)
(preparative)
CyDye™.
.
7
3–11 NL, 3–10 NL, 3–10
4–7
3–5.6 NL, 5.3–6.5, 6.2–7.5,
6–11, 7–11 NL
3–6
4–8
8–16
25–60
25–150
40–240
10
13
26
11
3–11 NL, 3–10
4–7
6–11, 3–5.6 NL, 5.3–6.5,
6.2–7.5, 7 –11 NL
7–15
10–20
20–40
50–120
50–300
100–600
20
28
56
13
3–11 NL, 3–10 NL, 3–10
4–7
6–11 narrow and medium intervals†
10–20
15–30
30–60
50–240
75–450
150–900
25
38
76
18
3–11 NL, 3–10 NL, 3–10
4–7
6–11, 6–9, narrow and medium intervals§
20–40
30–60
60–120
100–500
150–900
300–1500
50
75
150
24
3–11NL, 3–10 NL, 3–10
4–7, 3–7 NL
6–9, narrow and medium intervals§
30–60
45–90
80–200
200–600
200–1300
400–2000
100
150
300
* When using cup loading, an increased sample concentration will lead to an increased risk of protein precipitation in the sample cup.
A maximum concentration of 150 µg protein/150 µl sample solution (150 µl is the volume of the cup) is recommended. This is a
general recommendation, which will function for most samples, but the maximum concentration usable varies greatly between
sample types.
For larger sample loads, rehydration loading is recommended.
†
Immobiline DryStrip gels, pH intervals: 3–5.6 NL,5.3–6.5, 6.2–7.5, and 7–11 NL.
§
Immobiline DryStrip gels, pH intervals: 3–5.6 NL, 5.3–6.5, 6.2–7.5, 7–11 NL, 3.5–4.5, 4.0–5.0, 4.5–5.5, 5.0–6.0, and 5.5–6.7.
2.6 Immobiline DryStrip gel rehydration solutions
Immobiline DryStrip gels must be rehydrated prior to IEF. They should be rehydrated in the Immobiline DryStrip IPGbox
when the Manifold is used with Ettan IPGphor 3 and also when Multiphor II Electrophoresis System is used. When
using Ettan IPGphor 3 and standard Strip Holders, the strips should be rehydrated in the Strip Holders themselves.
Rehydration solution, which may or may not include the sample, is applied to the reservoir channels of the Immobiline
DryStrip IPGbox or Strip Holder, and then the Immobiline DryStrip gels are soaked individually.
There are two general rehydration methods: (1) passive rehydration, in which no electric field is applied during the
process, and (2) active rehydration, which is rehydration under low voltage (20–120 V). Active rehydration can facilitate
the entry into the strip of high-molecular-weight proteins (70). Passive rehydration can be done in the Strip Holder or
IPGbox, but active rehydration can be done only in the Strip Holder. Procedures for using both Strip Holders and the
IPGbox are described below, in section 2.7.
The IPGbox, a separate product, is required for proper strip rehydration when using the Manifold. The channel
in the Manifold is too wide to ensure proper absorption of the required volumes of rehydration solution.
The lid of IPG box prevents the strips from drying during rehydration thus eliminating the possibility of urea
crystallization and the need for Immobiline DryStrip Cover Fluid.
80-6429-60 AD 53
2.6.1 Components of rehydration solution
The choice of the most appropriate rehydration solution for the sample will depend on its specific protein solubility
requirements. A typical solution generally contains urea, nonionic or zwitterionic detergent, DeStreak Reagent or DTT,
the appropriate Pharmalyte or IPG Buffer (all available from GE Healthcare), and a tracking dye. The sample may also be
included. The role of each component is described below, as well as the recommended concentration range.
Urea solubilizes and denatures proteins, unfolding them to expose internal ionizable amino acids. Commonly,
8 M urea is used, but the concentration can be increased to 9 or 9.8 M if necessary for complete sample
solubilization.
Thiourea, in addition to urea, can be used to further improve protein solubilization, particularly for hydrophobic
proteins (10, 16, 55–57). When using both, the recommended concentration of urea is 7 M and that of thiourea 2 M.
Detergent solubilizes hydrophobic proteins and minimizes protein aggregation. The detergent must have zero net
charge—use only nonionic or zwitterionic detergents. CHAPS, Triton X-100, or NP-40 in the range of 0.5 to 4% are most
commonly used.
DeStreak Reagent overcomes the problems of streaking that commonly occur due to reoxidation when running gels
that contain basic regions above pH 7. The reagent stabilizes thiol groups such as disulfides, thus reducing streaking
and extra spots caused by various oxidation stages of proteins (62). See section 2.6.2 for more information and a
protocol for use of this reagent.
DeStreak Rehydration Solution contains DeStreak Reagent, as described above. The Rehydration Solution also
contains optimized concentrations of urea, thiourea, and CHAPS, and is ready for use after addition of the
appropriate IPG Buffer.
IPG Buffer or Pharmalyte (carrier ampholyte mixtures) improves separations, particularly with high sample loads.
Carrier ampholyte mixtures enhance protein solubility and produce more uniform conductivity across the pH gradient
without disturbing IEF or affecting the shape of the gradient. IPG Buffers are carrier ampholyte mixtures specially
formulated not to interfere with silver staining following 2-D electrophoresis. Select an IPG Buffer with the same pH
interval as the Immobiline DryStrip to be rehydrated (Table 17).
The advantages of increased concentration of IPG Buffer/Pharmalyte are:
• Improved sample solubilization
• Increased tolerance to salt in sample
• More even conductivity in the gel
Higher concentrations of IPG Buffer/Pharmalyte will limit the voltage usable during IEF and increase the time
required for the focusing step.
Silver staining may require a prolonged fixing step to wash out carrier ampholyte that may cause staining
background near the ion front of the second-dimension gel.
IPG Buffer or Pharmalyte can be included in the stock rehydration solution or added just prior to use. The
carrier ampholytes are included in the stock solution when multiple Immobiline DryStrip gels of the same pH
range are to be used. Carrier ampholytes are added to single aliquots of the stock solution when the same
stock solution will be used with different pH range Immobiline DryStrip gels.
Tracking dye (bromophenol blue) allows IEF progress to be monitored during the protocol. If the tracking dye
does not migrate toward the anode, no current is flowing. Note: the dye migrates to the end of the strip well
before the sample is focused!
Sample can be applied by including it in the rehydration solution. Up to 1 mg of sample per strip (dependent on the
length of the strip and the pH range) can be diluted or dissolved in rehydration solution prior to IEF. The amount
of sample required is dictated in part by the detection or visualization method used. For example, radiolabeling
requires a very small amount of sample whereas Coomassie blue staining requires larger sample amounts.
54 80-6429-60 AD
2.6.2 Using DeStreak Rehydration Solution
Nonspecific oxidation of protein thiol groups is a common problem during 2-D electrophoresis, especially at pH > 7. In
the resulting protein map, this problem manifests as horizontal streaks and extra spots.
DeStreak Reagent and DeStreak Rehydration Solution act to transfer thiol groups in proteins to stable disulfide groups,
thus preventing nonspecific oxidation. This will reduce streaking between spots in the protein map, especially in
the pH range 7–11, and also simplify the spot pattern by reducing the number of spots caused by protein oxidation
(compare A and B in Figure 25).
When rehydrating Immobiline DryStrip gels with solutions other than DeStreak Rehydration Solution, DeStreak
Reagent can be added to the sample solution to stabilize thiol groups and prevent nonspecific oxidation. DeStreak
Reagent is compatible with most sample solutions as long as they do not contain more than 20 mM reducing agents,
such as dithiothreitol (DTT), β-mercaptoethanol, or tris(2-carboxyethyl)phosphine (TCEP).
A)
B)
Fig 25. A. Without DeStreak Rehydration Solution. Anodic cup loading. Sample (100 µl, 0.8 mg/ml mouse liver protein) contained 8 M
urea, 0.5% CHAPS, 1% Pharmalyte pH 8–10.5, and 10 mM DTT. Immobiline DryStrip gel, pH 6–9, 24 cm, rehydrated in 1% IPG Buffer pH
6–11 with 8 M urea, 0.5% CHAPS, and 10 mM DTT. B. With DeStreak Rehydration Solution. Anodic cup loading. Sample (100 µl, 0.8 mg/ml
mouse liver protein) contained 8 M urea, 0.5% CHAPS, 1% Pharmalyte pH 8–10.5, and 10 mM DTT. Immobiline DryStrip gel, pH 6–9, 24
cm, rehydrated in DeStreak Rehydration Solution and 1% IPG Buffer pH 6–11.
Protocol: DeStreak Rehydration Solution
Reagents supplied
DeStreak Rehydration Solution (5 × 3 ml).
Required but not provided
Sample buffer containing reducing agents (up to 20 mM), such as DTT, β-mercaptoethanol, or tris(2-carboxy-ethyl)
phosphine (TCEP); IPG Buffer or Pharmalyte.
Preliminary steps
Before use, equilibrate DeStreak Rehydration Solution at room temperature for 30 min. Shake the bottle to dissolve the
urea crystals.
Sample preparation
1. Prepare the protein extract in sample buffer containing reducing agents, such as dithiothreitol (DTT),
β-mercaptoethanol, or tris(2-carboxyethyl)phosphine (TCEP), at a concentration of 20 mM.
Note: Using cup application, the sample solution may contain up to 1 mg of protein/ml. Using anodic paper-bridge
loading, higher concentrations can be used.
Preparation of DeStreak Rehydration Solution
1. DeStreak Rehydration Solution is supplied without IPG Buffer. Before use, add 15 µl (0.5%) or 60 µl (2.0%) of the
appropriate IPG Buffer or Pharmalyte to 3 ml of DeStreak Rehydration Solution.
Use 0.5% IPG Buffer in the DeStreak Rehydration Solution when:
•
IPGphor standard Strip Holder is used for the first dimension.
80-6429-60 AD 55
•
Horizontal gels are used in the second dimension.
•
10 kV is used in the Manifold.
•
Immobiline DryStrip 7–11 NL and 3–11 NL are used. This will give a high voltage and a short run time in hours,
both of which are essential for streak-free results.
Use 2.0% IPG Buffer in the DeStreak Rehydration Solution in cases where the highest solubility of proteins and
stability against salt are needed. Under these conditions, conductivity will be higher and the highest voltage
may not be reached.
Note: Select an IPG Buffer with the same pH interval as the Immobiline DryStrip being rehydrated. See Table
17 for buffers.
Table 17. Immobiline DryStrips and IPG buffers.
Immobiline DryStrip
IPG Buffer
pH 3.5–4.5, 3–5.6 NL
3.5–5.0
pH 3–7 NL, 4–7 4–7
pH 3–10 3–10
pH 3–10 NL 3–10 NL
pH 3–11 NL
3–11 NL
pH 5.3–6.5
5.5–6.7
pH 6–9, 6–11, 6.2–7.5 6–11
pH 7–11 NL 7–11 NL
Rehydration of Immobiline DryStrips
1. Pipette the appropriate volume of prepared DeStreak Rehydration Solution into the Reswell Tray or into the regular
Strip Holder as indicated in Table 18. Distribute the solution evenly over the channel length.
2. Carefully remove the cover foil from the Immobiline DryStrip, starting from the anodic end (+ end).
3. Carefully place the Immobiline DryStrip into the tray/holder channel, gel-side down. Take care to distribute the
rehydration solution evenly under the strip. To help coat the entire gel, gently lift and lower the strip and slide it
back and forth along the surface of the solution. Be careful not to trap bubbles under the Immobiline DryStrip gel.
4. Close the lid to the IPGbox..
5. Rehydrate for 10–20 h.
Sample application
1. Load the sample either in the rehydration solution or after rehydration using a sample cup or anodic paper bridge.
•
With acidic pH intervals (3.5–4.5, and 3–5.6 NL), we recommend rehydration loading or cathodic
sample cup application. Use up to 20 mM reducing agent per 100 µl of sample.
•
With neutral (5.3–6.5, and 4–7) and wide (3–10) pH intervals, all sample application methods can be
used, but sample-specific limitations may exist. Use up to 10 mM reducing agent per 100 µl of sample.
•
With basic Immobiline DryStrip (pH intervals 6.2–7.5, 6–9, 6–11, and 7–11 NL), we recommend anodic cup
application or anodic paper-bridge loading. Use up to 20 mM reducing agent per 100 µl of sample.
•
Using rehydration loading on basic strips, the sample (in rehydration solution) may contain up to 1 mM
reducing agent. This reducing power will be consumed during the rehydration step and early start of the run,
and thiols will be transferred to disulfides during the run.
56 80-6429-60 AD
Table 18. Rehydration solution volume per Immobiline DryStrip—Ettan IPGphor 3 protocol.
Immobiline DryStrip
gel length (cm)
Total volume per strip*.
(µl)
7
125
11
200
13
250
18
340
24
450
* Including sample, if applied.
Run
Run the gels according to the instructions included with the Immobiline DryStrip package.
Protocol: DeStreak Reagent
Reagents supplied
DeStreak Reagent (1 ml).
Required but not provided
Rehydration solution without reducing agents; sample buffer containing reducing agents (up to 20 mM), such as DTT,
β-mercaptoethanol, or tris(2-carboxyethyl)phosphine (TCEP).
1. Prepare DeStreak Reagent for use by adding 12 µl (15 mg) of DeStreak Reagent per ml of rehydration solution
containing no reducing agents.
2. Follow the steps for sample preparation, application, and gel run as previously provided for DeStreak Rehydration
Solution.
2.6.3 Preparation of other rehydration solutions
Typical compositions of rehydration solutions are given in appendix I, solutions C and D.
2.7 Immobiline DryStrip Gel rehydration
using accessories
This section includes protocols for use of the Strip Holder, IPGbox, and Manifold. It covers the following scenarios:
• Using Strip Holders for rehydration loading (sample included) or sample loading after gel rehydration.
• Using IPGbox prior to using the Manifold (sample added prior to reswelling or after reswelling using cup or paperbridge loading).
For rehydration sample loading, the Immobiline DryStrip gel must be rehydrated in the Immobiline DryStrip IPGbox or in
the standard Strip Holder. Mix the sample with rehydration solution (see section 2.5 for recommended sample loads).
When the Immobiline DryStrip gels are rehydrated with the sample proteins, sample cups are not used. This approach
is referred to as passive rehydration. In some cases, rehydration under voltage, referred to as active rehydration,
might be preferred. Rehydration under low voltage (20–120 V) facilitates the entry of high-molecular-weight proteins
(70). Active rehydration is possible only in the Strip Holder.
Large sample volumes and large protein amounts can be applied using paper-bridge loading (Manifold only). For
example, for basic proteins, a paper pad (paper bridge) is soaked with sample and placed between the anodic end
of the Immobiline DryStrip gel and the electrode (375–500 µl sample can be applied using the paper-bridge pads
supplied with the Manifold). Solutions containing up to 5 mg of protein have been loaded on an 18-cm narrow–pH
range Immobiline DryStrip gel (74).
A standard paper electrode pad between the paper bridge and the electrode improves sample transfer and
gel results.
80-6429-60 AD 57
The rehydrated Immobiline DryStrip gel is first positioned in the bottom of the Manifold channel, gel side up. Then
the paper bridge with sample is positioned, followed by a paper wick. With anodic application the anode electrode is
positioned as far out as possible in the electrode assembly, while the cathode electrode is positioned close to the end
of the Immobiline DryStrip gel to ensure good contact between the paper wick and Immobiline DryStrip gel.
The application point (anodic or cathodic) is an important factor for obtaining good results.
A single paper bridge can be used with the 24-cm gel strip. If so desired, a paper bridge can be used on both
ends of all other strips at one time.
Protocol: Using the Strip Holder for gel rehydration
IPGphor fixed-length Strip Holders allow IPG strips to be rehydrated and samples loaded in one step before proceeding
automatically to perform the IEF separation. The IPG strips are 3 mm wide and 0.5 mm thick after rehydration.
This protocol applies for both in-gel sample rehydration and sample application after gel rehydration. In the latter
case, see Note C below.
1. Prepare the Strip Holder(s)
Select the Strip Holder(s) corresponding to the Immobiline DryStrip gel length chosen for the experiment.
Handle the ceramic Strip Holders with care.
It is essential to wash each Strip Holder with detergent to remove residual protein. Use a neutral pH
detergent, such as the Strip Holder Cleaning Solution, to remove residual protein from the Strip Holders. Strip
Holder Cleaning Solution has been specifically formulated to remove protein deposits and will not damage
the Strip Holder. Strip Holder Cleaning Solution is available in 950-ml bottles (see ordering information).
1. Clean Strip Holders after each first-dimension IEF run. Do not let solutions dry in the Strip Holder. Cleaning may be
more effective if the Strip Holders are first soaked for a few hours or overnight in a solution of 2–5% Strip Holder
Cleaning Solution in water. First rinse off the Strip Holder to remove any residual DryStrip Cover Fluid.
2. Squeeze a few drops of Strip Holder Cleaning Solution into the Strip Holder channel. Use a toothbrush and
vigorous agitation to clean the Strip Holder.
3.
Rinse well with distilled or deionized water. Thoroughly air-dry the Strip Holders or dry well with a lint-free tissue
prior to use.
Recalcitrant or dried-on protein deposits may be removed with hot (up to 95 ºC) 1% (w/v) SDS. Add 1% (w/w) DTT
for complete removal of sticky proteins. Rinse thoroughly with distilled or deionized water after cleaning.
Handle clean Strip Holders with gloves to avoid contaminating them.
Strip Holders may be baked, boiled, or autoclaved. DO NOT EXPOSE THEM TO STRONG ACIDS OR BASES,
INCLUDING ALKALINE DETERGENTS.
The Strip Holder must be completely dry before use.
2.
Apply the rehydration solution
Pipette the appropriate volume of rehydration solution into each Strip Holder as indicated in Figure 26. Deliver the
solution slowly as a stripe of liquid between the two electrodes, away from the sample application wells (Fig 26).
Remove any large bubbles.
For a typical composition of rehydration solution, see appendix I, solution C. If in-gel sample rehydration is
desired, add the appropriate amount of sample to the rehydration solution. Recommended loads are shown in
Table 16 (section 2.5).
To ensure complete sample uptake, do not exceed the recommended volume of rehydration solution, see
Table 18 (section 2.6.2).
3.
Position the Immobiline DryStrip gel
Remove the protective cover foil from the Immobiline DryStrip gel starting at the acidic (+) end. Removal from the
acidic end prevents damage to the basic end of the gel, which is generally softer. Using forceps, position the
Immobiline DryStrip gel with the gel side down and the anodic (+) end of the strip directed toward the pointed end
of the Strip Holder (Fig 27). Acidic end first, lower the gel onto the solution. To help coat the entire strip, gently
lift and lower the strip and slide it back and forth along the surface of the solution, tilting the Strip Holder slightly
as required to ensure complete and even wetting. Finally, lower the cathodic end of the Immobiline DryStrip gel
into the channel, making sure that the gel contacts the Strip Holder electrodes at each end. (The gel can be
visually identified once the rehydration solution begins to enter the gel.) Be careful not to trap air bubbles under
the Immobiline DryStrip gel.
58 80-6429-60 AD
4.
Apply Immobiline DryStrip Cover Fluid
Apply Immobiline DryStrip Cover Fluid to minimize evaporation and thus prevent urea crystallization. Pipette the
fluid dropwise into one end of the Strip Holder until one half of the Immobiline DryStrip gel is covered. Then pipette
the fluid dropwise into the other end of the Strip Holder, adding fluid until the entire gel is covered.
5. Place the cover on the Strip Holder
Pressure blocks on the underside of the cover ensure that the Immobiline DryStrip gel maintains good contact
with the electrodes as the gel swells.
6.
Allow the Immobiline DryStrip gel to rehydrate
Rehydration can proceed on the bench top or on the Ettan IPGphor 3 platform. Ensure that the Strip Holder is on
a level surface. A minimum of 10 h is required for rehydration; overnight is recommended. The rehydration period
can be programmed as the first step of an Ettan IPGphor 3 protocol. This is especially convenient if temperature
control during rehydration is a concern.
Active rehydration (20–120 V) can also be performed if sample is included.
a)
b)
c)
Fig 26. Applying rehydration solution into the Strip Holder. (a). Positioning the Immobiline DryStrip gel (b, c). Positioning the Immobiline
DryStrip gel.
A. Rehydration loading
A discussion of the advantages of rehydration loading can be found in section 2.4.
B. Optional: Apply electrode pads
During IEF, the transport of ions, proteins, and IPG Buffer between the electrodes is accompanied by transport
of water. For large sample loads and when using narrow-pH-range Immobiline DryStrip gels, better results are
obtained by applying damp paper pads between the Immobiline DryStrip gel and each Strip Holder electrode
following rehydration but before IEF, in order to absorb excess water.
1.
Prepare electrode pads
Use the paper wicks (accessory to the Manifold) or cut two 3-mm-wide electrode pads from a paper IEF electrode strip.
Place on a clean, flat surface such as a glass plate and soak with deionized water. Remove excess water by
blotting with tissue paper.
Electrode pads must be damp, not wet.
2. Position electrode pads
Using forceps, lift one end of the rehydrated Immobiline DryStrip gel. Position an electrode pad over the electrode,
then lower the gel strip back into place. Repeat at the other end.
Additional DryStrip Cover Fluid may need to be added to ensure that the strip is still adequately covered.
80-6429-60 AD 59
C. Apply sample after gel rehydration
If the sample was not applied as a part of the rehydration solution, it can be applied immediately prior to IEF.
1. Prepare sample
Prepare the sample in a solution similar in composition to the rehydration solution used.
2.
Apply sample
Pipette the sample into either or both of the lateral wells at either end of the Strip Holder (Fig 27). Introduce the
sample below the Immobiline DryStrip Cover Fluid.
Up to 7.5 µl of sample solution can be added to each side (i.e. 15 µl per well or 30 µl total if both sides of both
wells are used).
The Immobiline DryStrip gel backing is impermeable; do not apply the sample to the back of the strip.
Replace cover on Strip Holder.
Refer to Table 18, section 2.6.2 for rehydration solution volume per Immobiline DryStrip.
Fig 27. Applying sample after gel rehydration.
Protocol: Using IPGbox for Rehydration
If the Manifold is used, Immobiline DryStrip gels must be rehydrated prior to IEF in the Immobiline DryStrip IPGbox.
Rehydration can take place with or without the sample included.
Do not use the Manifold for rehydration.
Each tray has 12 independent reservoir channels that can each hold a single Immobiline DryStrip gel up to 24 cm
long. Separate channels allow the rehydration of individual gel strips with no danger of spillover into adjacent lanes.
1 Select and prepare a rehydration solution, see Section 2.6.1.
2 Place the IPGbox on a levelled table and place a Reswell Tray in the IPGbox. Ensure that the Reswell Tray and the
IPGbox Insert are clean and dry. If required, wipe the IPGbox Insert with a soft tissue moistened with 70% ethanol.
3 Pipette the appropriate volume of selected rehydration solution (and sample if applied) evenly over the slots
corresponding to the length of the DryStrip gels. See Table 18 for rehydration solution volumes. Strip lengths are
measured from the straight ended part of the wells of the Reswell Tray and marked with lines on the top of the
Reswell Tray.
Note: For complete sample uptake, do not apply excess rehydration solution.
4 Carefully pull off the cover film from Immobiline DryStrip gel and place Immobiline DryStrip gel into the slot, gel-side
down. Distribute the rehydration solution evenly under the strip. Gently lift and lower the strip and slide it back and
forth along the surface of the solution to get complete and even wetting of the entire gel.
Note: Ensure that no bubbles are trapped under the Immobiline DryStrip gel.
60 80-6429-60 AD
Fig 28. Example of IPG strips in a Reswell Tray.
5 Gently close the lid of the IPGbox and allow the Immobiline DryStrip gels to rehydrate at room temperature for
10-24 hours (overnight is recommended).
6 Use the Immobiline DryStrip gels within 20 minutes of opening the IPGbox. Discard the used Reswell Tray.
Note: Do not store the Reswell Tray in IPGbox. Long time pressure of a Reswell Tray can reduce the life span of the
IPGbox Insert.
Protocol: Preparing the Manifold
1.
Clean and dry the IPGphor 3 bed before placing the Manifold tray on the unit. Position the Manifold on the
IPGphor 3 platform. The small T-shaped protrusion fits into a cutout section of the IPGphor bed near the lid hinge
(Fig 29). Ensure that the Manifold is level by placing the round spirit level on the center of the Manifold tray after it
is placed on the Ettan IPGphor 3 unit. Adjust leveling feet if necessary.
Important! Before proceeding, make sure the Ettan IPGphor 3 unit is placed on a level surface.
Important! If using the original Ettan IPGphor, ensure that the three foam pads have been removed from the
lid of the unit. (This step is not necessary if using Ettan IPGphor 3.)
2.
Measure out 108 ml of Immobiline DryStrip Cover Fluid (even if fewer than 12 strips will be loaded into the Manifold).
Add the cover fluid evenly between the 12 Manifold channels. Transfer the strips to the Ettan IPGphor Manifold.
Place the strips under the cover fluid, gel side up in the tray with the anodic (+) end of the IPG strip oriented
toward the anodic side of the instrument. Position the strip to rest on the appropriate mark etched into the
bottom of the Manifold channel (the end of the gel, not the end of the plastic, should align with the etched mark).
Center the strip down the length of the Manifold channel. Protrusions along the sides guide the strip approximately
straight, although some manual adjustment of the strip may be necessary (Fig 30).
Note: If cathodic cup loading is going to be used, the strips should be placed such that the anodic end of the
strips is 3–4 cm beyond the etched placement mark.
3.
If performing cup loading, place a strip of cups in the appropriate position (Fig 31), for example ~1 cm from the
end of the gel portion of the IPG strip. Do NOT place the cup with the feet over a center protrusion. Push the cups
into the channels with gloved fingers, starting at one end and working toward the other. Align the insertion tool
over the cups and push down to ensure that the feet of the cups are properly seated at the bottom of the channel
(wiggle the tool gently while pushing down in order to ensure that the cups are seated as far down as they will go).
Take care not to move the cups while removing the insertion tool. Ensure that the cups are filled with cover fluid.
If desired, test for leakage by adding some colored sample buffer (without sample). If no leaks are detected,
pipette the colored liquid back out again.
Cups must not straddle the centering protrusions on the bottom of the channels.
4.
Count out the appropriate number of precut paper wicks. Two wicks per strip are required. Separate the wicks
from each other. Add 150 µl of distilled water to each wick. Place the wicks on the IPG strips such that one end
of the wick overlaps the end of the gel on the IPG strip (Fig 32). For gradients with pH above 9, add 150 µl DeStreak
rehydration solution to the cathodic wick. The electrode must contact the wick. With the electrode cams in the open
position, place the electrode assembly on top of all the wicks. Swivel the cams into the closed position under the
external lip of the tray. The electrodes should not be moved while the cams are in the closed position (Fig 33).
5.
Briefly centrifuge the protein sample (e.g. at top speed in a microcentrifuge) prior to loading to remove insoluble
material and particulate matter. These materials could impede sample entry and result in vertical streaks in the
second-dimension gel. Load samples into the sample cups. A maximum of 150 µl of sample may be placed in
these cups. Check to make sure that there is cover fluid over the samples. When the cups are initially placed on
the Manifold, cover fluid will flow into the cups as they are seated. When sample is introduced into the cups, the
sample will sink to the bottom of the cup and contact the IPG strip.
80-6429-60 AD 61
Note: For basic IPG strips, superior focusing patterns are generally obtained when the sample cup is placed as
close to the anodic (+) electrode as possible.
6.
Close the Ettan IPGphor 3 lid. Program the Ettan IPGphor 3 with the desired run parameters. Ramping the voltage
slowly while the sample is entering the IPG strip will improve results. See section 2.8 for further discussion on this
topic. Optimal ramp, voltages and times, or Vhr (volt-hours) totals must be determined empirically for each
sample type. Focusing after sample cup application frequently requires fewer Vhr than in-gel sample rehydration
loading methods, particularly on basic pH-range strips.
Incorrect cup position
Correct cup position
Fig 29. Manifold placement on Ettan IPGphor 3.
Fig 31. Sample cup positioning details.
Note: Cups must not straddle the centering protrusions on the
bottom of the channels.
24 cm
18 cm
13 cm
11 cm
Fig 32. Correct placement of paper wicks.
7 cm
Fig 30. Placement of IPG strips in Manifold channels.
Note: If cathodic cup loading is going to be used, the strips
should be placed such that the anodic end of the strips is 3–4 cm
beyond the etched placement mark.
62 80-6429-60 AD
Fig 33. Placement of electrode on paper wicks. Cams are in the
open position.
2.8 Isoelectric focusing guidelines—
Ettan IPGphor 3 System
IEF using the Ettan IPGphor 3 Isoelectric Focusing System is conducted at very high voltages (up to 10 000 V,
depending on the length of the DryStrip used) and very low currents (typically less than 50 µA per Immobiline DryStrip
gel) due to the low ionic strength within Immobiline DryStrip gels. During IEF, the current decreases while the voltage
increases as proteins and other charged components migrate to their equilibrium positions. A typical IEF protocol
generally proceeds through a series of voltage steps that begins at a relatively low value. Voltage is gradually
increased to the final desired focusing voltage, which is held for several hours. A low initial voltage minimizes sample
aggregation and allows the parallel separation of samples with differing salt concentrations. A gradual increase in
voltage is particularly advisable for higher protein loads (100 µg or more per Immobiline DryStrip gel).
Many factors affect the amount of time required for complete focusing, and each specific set of conditions (e.g.
sample and rehydration solution composition, Immobiline DryStrip gel length, and pH gradient) requires empirical
determination for optimal results. An approximate time for complete focusing is given in the example protocols
provided in Table 19. Factors that increase the required focusing time include residual ions, which must move to the
ends of the Immobiline DryStrip gels before protein focusing can occur, and the presence of IPG Buffers or Pharmalyte,
which contributes to the ionic strength of the electrophoresis medium. A higher IPG Buffer concentration increases
the conductivity of the Immobiline DryStrip gel, resulting in a lower final voltage when the system is limited by the
maximum current setting.
Longer focusing times may therefore be required at IPG Buffer/Pharmalyte concentrations higher than 0.5%.
For higher protein loads (up to 1 mg or more) the final focusing step of each protocol can be extended if
necessary by an additional 20% of the total recommended Volt-hour value.
Exceeding the current limit of 50 µA/Immobiline DryStrip gel is not recommended, as this may result in
excessive heat generation and may damage the Immobiline DryStrip gel and/or Manifold or Strip Holder.
Under extreme circumstances, the Immobiline DryStrip gel may burn.
Overfocusing can sometimes occur on longer runs and may contribute to horizontal streaking, which will be
visible in the 2-D gel result (see also chapter 7, Troubleshooting).
2.8.1 Protocol examples—Ettan IPGphor 3 Isoelectric Focusing System
These protocols are suitable for first-dimension isoelectric focusing of protein samples prepared in rehydration
solution in typical analytical quantities (1–100 µg).
The protocols are optimized for a rehydration solution containing 0.5% IPG Buffer or Pharmalyte. The recommended
current limit is 50 µA/Immobiline DryStrip gel. Recommended focusing times are given, but the optimal length of time
will depend on the nature of the sample, the amount of protein, and the method of sample application.
Please refer to the Ettan IPGphor 3 user manual for instructions on how to program a protocol.
2.8.2 Running an Ettan IPGphor 3 protocol
Ensure that the Strip Holders are properly positioned on the Ettan IPGphor 3 platform. Use the guide marks along
the sides of the platform to position each Strip Holder and check that the pointed end of the Strip Holder is over the
anode (pointing to the back of the unit) and the blunt end is over the cathode. (Please refer to the Ettan IPGphor 3
user manual for complete details.) Check that both external electrode contacts on the underside of each Strip
Holder make metal-to-metal contact with the platform.
Before closing the safety lid, insert the lid adaptor (an accessory included with IPGphor 3) such that the pressure
pads press gently against the cover of each Strip Holder to ensure contact between the electrodes and the
electrode areas. Begin IEF.
80-6429-60 AD 63
As isoelectric focusing proceeds, the bromophenol blue tracking dye migrates toward the anode. Note that
the dye front leaves the Immobiline DryStrip gel well before focusing is complete, so clearing of the dye is
no indication that the sample is focused. If the dye does not migrate, no current is flowing. If this occurs,
check the contact between the external face of the Strip Holder electrodes and the electrode areas on the
instrument, and between the rehydrated gel and the internal face of the electrodes. Table 19 lists guidelines
for running Immobiline DryStrip gels on Ettan IPGphor 3.
It is possible that the programmed maximum voltage will not be reached when using shorter Immobiline
DryStrip gels or with samples having high conductivity.
The final step of focusing should be run in volt-hours to ensure reproducibility from run to run.
The following protocols are suitable for first-dimension isoelectric focusing of proteins run on Ettan IPGphor 3
Isoelectric Focusing Unit.
Preparative sample loads often increase the electroosmotic pumping of water. Excess free water on the
gel surface contributes to streaky results and should be absorbed with electrode pads. This technique is
standard when using the Ettan IPGphor 3 Manifold; for standard Strip Holders this technique is described in
section 2.7.
The focusing times below are guidelines only, based on well-prepared samples. Times may vary with the
nature of the sample and how the sample is applied. Using crude samples with high protein and salt content
or using paper-bridge loading, the run time in total kiloVolt-hours should be increased by 10%.
For Immobiline Dry Strip pH 6.2–7.5, 6–9, 6–11, and 7–11 NL, loading the sample anodically in a sample cup is
recommended. For preparative sample loads with these basic strips, paper-bridge loading is recommended.
If using the Manifold and 18- and 24-cm strips, the maximum voltage is 10 000 V. With these two strip lengths
and standard Strip Holders, the maximum allowed voltage is 8000 V. With all other strips and regardless of
whether the Manifold is being used, the maximum voltage is 8000 V.
Table 19. Guidelines for running 7–24-cm Immobiline DryStrip gels on Ettan IPGphor 3 Isoelectric Focusing Unit. Running conditions:
Temperature 20 °C; current 50 µA per strip except where noted. See footnotes for information specific to the different strip lengths.
7-cm strips
pH Voltage mode intervals Voltage (V) Time (h:min)
kVh.
3–11 NL 3–10 6–11 1 Step and Hold
300 2 Gradient 1000 3 Gradient 5000 4 Step and Hold 5000 Total 0:30
0:30 1:20 0:06–0:25 2:26–2:45 0.2
0.3
4.0
0.5–2.0
5.0–6.5
3–10 NL 4–7 3–5.6 NL 1 Step and Hold
300 2 Gradient 1000 3 Gradient 5000 4 Step and Hold 5000 Total 0:30
0:30 1:30 0:12–0:36 2:42–3:06 0.2
0.3
4.5
1.0–3.0
6.0–8.0
7–11 NL 1 Step and Hold
300 2 Gradient 1000 3 Gradient 5000 4 Step and Hold 5000 Total 0:30
1:00 1:30 0:20–0:55 3:20–3:55 0.2
0.7
4.5
1.6–4.6
7.0–10.0
5.3–6.5 6.2–7.5 1 Step and Hold
300 2 Gradient 1000 3 Gradient 5000 4 Step and Hold 5000 Total 1:00
1:00 2:30 0:45–1:30 5:15–6:00 0.2
0.7
7.5
3.6–7.6
12.0–16.0
64 80-6429-60 AD
Table 19. (continued)
11-cm strips
*
pH intervals Step Voltage mode 3–11 NL
3–10 6–11 Voltage (V) Time (h:min)
kVh.
1 Step and Hold
500 2 Gradient 1000 3 Gradient 6000 4 Step and Hold 6000 Total 1:00 1:00 2:00 0:10–0:40 4:10–4:40 0.5
0.8
7.0
0.7–3.7
9.0–12.0
4–7 3–5.6 NL 1 Step and Hold
500 2 Gradient 1000 3 Gradient 6000 4 Step and Hold 6000 Total 1:00 1:00 2:30 0:10–0:50 4:40–5:20 0.5
0.8
8.8
0.9–4.9
11.0–15.0
7–11 NL 1 Step and Hold
500 2 Gradient 1000 3 Gradient 6000 4 Step and Hold 6000 Total 1:00 1:00 2:30 0:50–1:40 5:20–6:10 0.5
0.8
8.8
4.9–9.9
15.0–20.0
5.3–6.5 6.2–7.5 1 Step and Hold* 500 2 Gradient 1000 3 Gradient 6000 4 Step and Hold 6000 Total 1:00*
1:00 3:00 2:40–3:50 7:40–8:50 0.5
0.8
10.5
16.2–23.2
28.0–35.0
To convert this to a convenient overnight run, extend Step 1 to 6 h (3 kVh) and reduce step 4 by 3 kVh.
13-cm strips
pH intervals Step Voltage mode 3–10 3–11 NL 6–11 Voltage (V) Time (h:min)
kVh.
1 Step and Hold
500 2 Gradient 1000 3 Gradient 8000 4 Step and Hold 8000 Total 1:00 1:00 2:30 0:10–0:30 4:40–5:00 0.5
0.8
11.3
1.4–4.4
14.0–17.0
3–10 NL 4–7 3–5.6 NL 1 Step and Hold
500 2 Gradient 1000 3 Gradient 8000 4 Step and Hold 8000 Total 1:00 1:00 2:30 0:25–0:55 4:55–5:25 0.5
0.8
11.3
3.4–7.4
16.0–20.0
7–11 NL 1 Step and Hold
500 2 Gradient 1000 3 Gradient 8000 4 Step and Hold 8000 Total 1:00 1:00 3:00 0:45–1:15 5:45–6:15 0.5
0.8
13.5
6.2–10.2
21.0–25.0
5.3–6.5 6.2–7.5 1 Step and Hold* 500 2 Gradient 1000 3 Gradient 8000 4 Step and Hold 8000 Total 1:00* 1:00 3:00 2:55–4:10 7:55–9:10* 0.5
0.8
13.5
23.2–33.2
38.0–48.0
* To convert this to a convenient overnight run, extend Step 1 to 6 h (3 kVh) and reduce step 4 by 3 kVh.
80-6429-60 AD 65
Table 19. (continued)
18-cm strips
Note: When using IPGphor Manifold and 10 kV, set current limit to 75 µA per strip and follow step 1, 2, 3b and 4b. Using
IPGphor Regular Strip Holder or Cup Loading Strip Holder with the 18- and 24-cm strips, the maximum allowed
voltage is 8000 V and current 50 µA per strip. Follow step: 1, 2, 3a, 4a.
pH intervals Step Voltage mode Voltage (V) Time (h:min)
Volt-hours.
kVh
3–10 3–11 NL 6–11 1 Step and Hold* 500 1:00 (8:00)*
2 Gradient 1000 1:00
8000 3:00 3a Gradient†
8000 0:46–1:30 4a Step and Hold† 10000 3:00 3b Gradient‡ 10000 0:20–0:55 4b Step and Hold‡ Total 0.5
0.8
13.5
6.2–12.2
16.5
3.2–9.2
21.0–27.0
3–10 NL 4–7 3–5.6 NL 500 1:00 (8:00)* 1 Step and Hold‡ 2 Gradient 1000 1:00 †
8000 3:00 3a Gradient 8000 1:30–2:40 4a Step and Hold† 10000 3:00 3b Gradient‡ 10000 0:55–1:50 4b Step and Hold‡ Total 0.5
0.8
13.5
12.2–21.2
16.5
9.2–18.2
27.0–36.0
6–9 7–11 NL 1 Step and Hold* 500 1:00 (8:00)* 2 Gradient 1000 1:00 8000 3:00 3a Gradient†
8000 3:10–4:30 4a Step and Hold† ‡
10000 3:00 3b Gradient 10000 2:15–3:15 4b Step and Hold‡
Total 0.5
0.8
13.5
25.2–35.2
16.5
22.2–32.2
40.0–50.0
5.3–6.5 6.2–7.5 1 Step and Hold* 500 2:00 (3:00)*
2 Gradient
1000 2:00 8000 3:00 3a Gradient† 8000 6:45–8:40 4a Step and Hold† 10000 3:00 3b Gradient‡
10000 5:05–6:35 4b Step and Hold‡ Total 1.0
1.5
13.5
54.0–69.0
16.5
51.0–66.0
70.0–85.0
* When a more convenient overnight run of 15 to 17 h is desired, the time in step 1 can be extended up to recommended value
in brackets. Using this option, step 4 can be reduced with the added kVh in step 1, to reach the specified total kVh.
Follow steps 1, 2, 3a and 4a when using IPGphor Regular Strip Holder or Cup Loading Strip Holder.
†
Follow steps 1, 2, 3b and 4b when using IPGphor Cup Loading Manifold.
‡
66 80-6429-60 AD
Table 19. (continued)
24-cm strips
Note: When using IPGphor Manifold and 10 kV, set current limit to 75 µA per strip and follow step 1, 2, 3b and 4b. Using
IPGphor Regular Strip Holder or Cup Loading Strip Holder with the 18- and 24-cm strips, the maximum allowed
voltage is 8000 V and current 50 µA per strip. Follow step: 1, 2, 3a, 4a.
pH intervals Step Voltage mode Voltage (V) Time (h:min)
kVh.
3–11 NL 3–10 1 Step and Hold* 500 1:00 (8:00)*
2 Gradient 1000 1:00 8000 3:00 3a Gradient† 8000 2:30–3:45 4a Step and Hold† 10000 3:00 3b Gradient‡ 10000 1:45–2:45 4b Step and Hold‡ Total 0.5
0.8
13.5
20.0–30.0
16.5
17.2–27.2
35.0–45.0
3–10 NL 3–7 NL 4–7 3–5.6 NL 1 Step and Hold* 500 1:00 (7:00)*
2 Gradient 1000 1:00 †
8000 3:00 3a Gradient 8000 3:45–5:36 4a Step and Hold† 10000 3:00 3b Gradient‡ 10000 2:45–4:15 4b Step and Hold‡ Total 0.5
0.8
13.5
30.0–45.0
16.5
27.2–42.2
45.0–60.0
6–9 7–11 NL 1 Step and Hold* 500 1:00 (5:00)* 2 Gradient 1000 1:00 8000 3:00 3a Gradient† 8000 5:36–8:45 4a Step and Hold†
‡
10000 3:00 3b Gradient 10000 4:15–6:45 4b Step and Hold‡ Total 0.5
0.8
13.5
45.0–70.0
16.5
42.2–67.2
60.0–85.0
3.5–4.5 1 Step and Hold* 500 2:00 (5:00)* 2 Gradient 1000 2:00 8000 3:00 3a Gradient† 8000 9:10–10:30 4a Step and Hold†
10000 3:00 3b Gradient‡ 10000 7:05–8:05 4b Step and Hold‡ Total 1.0
1.5
13.5
74.0–84.0
16.5
71.0–81.0
90.0–100.0
5.3–6.5 6.2–7.5 1 Step and Hold* 500 2:00 (5:00)* 2 Gradient 1000 2:00 8000 3:00 3a Gradient†
8000 9:19-10:30
4a Step and Hold† 10000 3:00 3b Gradient‡
10000 9:05–11:05 4b Step and Hold‡ Total 1.0
1.5
13.5
94.0–114.0
16.5
91.0–111.0
110.0–130.0
* When a more convenient overnight run of 15 to 17 h is desired, the time in step 1 can be prolonged to up to recommended values
in brackets. Using this option, step 4 can be reduced with the added kVh in step 1, to reach the specified total kVh.
Follow steps 1, 2, 3a and 4a when using IPGphor Regular Strip Holder or Cup Loading Strip Holder.
†
Follow steps 1, 2, 3b and 4b when using IPGphor Cup Loading Manifold.
‡
If using regular Strip Holders, active rehydration can be performed (if sample is included) by adding an
extra step at the beginning of the protocol (e.g. Voltage mode = 1 Step and Hold, Voltage = 30,
Step duration = 10:00, kVolt-hours = 0.3 kVh).
2.8.3 Preservation of focused Immobiline DryStrip gels
After IEF is complete, proceed to the second-dimension separation immediately or store the Immobiline DryStrip gels
at -60 °C or below. This can be conveniently done by placing the strips between plastic sheets, as suggested by Görg
et al. (3) or on glass plates covered in plastic wrap. Alternatively, the DryStrip gels can be stored in screw-cap tubes.
The 7-cm strips fit in disposable 15-ml conical tubes; 11-, 13-, and 18-cm strips fit in 25 × 200 mm screw cap culture
tubes; and 18- and 24-cm strips fit in Equilibration Tubes (see ordering information). The equilibration process is
discussed in chapter 3.
80-6429-60 AD 67
2.9 Troubleshooting
Table 20 lists possible problems that might be encountered during IEF and how to solve them, and Table 21
lists problems and solutions when using Ettan Manifold.
Table 20. Troubleshooting first-dimension IEF: Ettan IPGphor 3 Isoelectric Focusing System.
Symptom Possible solutions
Problems indicated by LCD messages
Lid open step 1, close to continue The safety lid is not properly closed. When the safety lid is open, the
system has an automatic voltage cutoff safety feature. In order for the
protocol to proceed, the safety lid must be closed.
Locked screen in edit mode Turn off the mains power switch to reset the instrument.
Blank display If no electrical components are functioning (e.g. HV lamp does not light
and the cooling fans are motionless), check the fuses in the mains
power module.
Diagnostic program indicates component failure Note the component that failed and press the START key to continue
through the diagnostic program. Call your local GE Healthcare sales
office for further information on how to remedy the failure.
Power delivery
Current too low or zero At least two of three pressure pads on the lid adaptor of IPGphor 3 under
the safety lid should press gently against the strip holders to ensure
electrical continuity between the strip holder electrodes and the electrode
areas on the platform.
The gel must be evenly and completely rehydrated to conduct current.
Make sure the proper amount of rehydration solution is applied to the
IPG strip holder and allow a minimum of 10 hours for rehydration.
Voltage limit not reached The ionic strength of the rehydration solution is too high; reduce the IPG
buffer concentration; use a mixed-bed ion-exchange resin to remove
ionic breakdown products of urea or other additives.
Desalt the sample or prepare the sample so that the salt concentration
is less than 10 mM.
Sparks or burning in strips Reduce the current limit. Do not exceed 50 µA per strip.
Prevent the IPG strip from drying out by always applying Immobiline
DryStrip Cover Fluid immediately after strip placement in rehydration buffer.
Ensure that the IPG strip is fully rehydrated along the entire length of
the strip. The IPG strip should be in complete contact with the correct
volume of rehydration solution. Remove any air bubbles trapped under
the IPG strip.
Desalt the sample or prepare the sample so that the salt concentration
is less than 10 mM. De-ionize additives to the rehydration solution.
Excessive charged material in the sample or rehydration buffer can lead
to electroendosmosis, which could dry out part of the strip, possibly
leading to arcing and burning in this region.
68 80-6429-60 AD
Table 21. Troubleshooting first-dimension IEF: Employing the Manifold.
Symptom
Possible cause
Remedy
Electrical continuity is impeded.
Current is too low or zero
Check the external electrode contacts: Ensure correct
placement of the electrode assemblies such that there is
metal-to-metal contact with the appropriate electrode
contact area.
Check the internal electrode contacts: The gel (which becomes
visible because of the dye in the rehydration solution) must
contact both electrodes in the Manifold through the paper
wicks and/or paper bridge parts.
Check that the IPG strip is fully rehydrated along its entire
length. Electrical contact at the electrodes is reduced by
incomplete rehydration.
Check that the paper wicks are present and properly positioned.
Voltage too low or does not Ettan IPGphor protocol settings reach maximum set value
are incorrect for the experiment.
Check that the current limit is properly set.
Check that the actual number of Immobiline DryStrip gels on
the Ettan IPGphor platform is the same as the number of gels
entered in the protocol.
Conductivity/ionic strength is too high. Prepare the sample to yield a salt concentration less than
10 mM. The recommended IPG Buffer concentration is 0.5%.
A maximum of 2% is advisable only if sample solubility is a
problem. High conductivity can also arise from the use of poor
quality urea or other denaturants. Urea is also prone to
decomposing to charged breakdown products.
Higher conductivity salts and ionic impurities in the sample
can raise the conductivity of the strip.
Shorter length IPG strips (e.g. 7 cm strips) will not reach 8000 V.
The distance between the electrodes is shorter so that the
voltage gradient (V/cm) required to reach the 50 µA current
limit is reached at a lower overall voltage.
Sample leaks from cup Incorrect cup placement.
Check that the feet of the cups are resting on the bottom of
the manifold channel.
Check for correct positioning of sample cup arms.
Check that the feet of the cups are not resting on a centering
protrusion in the channel.
Check that the strip is centered inside of the channel.
Incorrect strip placement. Sparking or burning in the Current limit setting is too high. Immobiline DryStrip gels
Do not exceed the maximum recommended setting of 50 µA
per Immobiline DryStrip gel.
Immobiline DryStrip gel is not fully rehydrated.
Ensure that the Immobiline DryStrip gels are rehydrated with a
sufficient volume of rehydration solution. Remove any large
bubbles trapped under the Immobiline DryStrip gel after
placing it on rehydration solution.
Always apply Immobiline DryStrip Cover Fluid to prevent
dehydration of rehydrated Immobiline DryStrip gels.
Immobiline DryStrip gels dried out during IEF. Immobiline DryStrips turn Immobiline DryStrip white and opaque gels dried out during IEF. after focusing
Always apply recommended amount of Immobiline DryStrip
Cover Fluid to prevent dehydration of rehydrated Immobiline
DryStrip gels.
Excess cover fluid added. Immobiline DryStrip Cover Fluid overflows from Manifold
Do not add more than the recommended volume. Ensure that
the outside rim of the tray does not have any oil on it.
80-6429-60 AD 69
70 80-6429-60 AD
3. Second-dimension SDS-PAGE using
vertical electrophoresis systems
3.0 Overview
After IEF, the second-dimension SDS-polyacrylamide gel electrophoresis (SDS-PAGE) can be performed on various
vertical or flatbed systems, depending on factors such as those discussed in “Equipment Choices” on pages 14–16.
SDS-PAGE consists of four steps:
1) Preparing the system for second-dimension electrophoresis
2) Equilibrating the Immobiline DryStrip gel(s) in SDS equilibration buffer
3) Placing the equilibrated Immobiline DryStrip gel on the SDS gel
4) Electrophoresis
The equilibration step is described first because it is a protocol common to all electrophoresis systems described in
this handbook. Gel preparation, Immobiline DryStrip gel placement, and electrophoresis protocols, on the other hand, are
specific to the orientation of the gel. Sections 3.3 and 3.4 describe these protocols as they apply to vertical systems;
sections 4.1 and 4.2 describe them as they apply to the flatbed Multiphor II Electrophoresis System.
3.1 Equilibrating Immobiline DryStrip gels
As mentioned in chapter 2, after IEF it is important to proceed immediately to gel equilibration, unless
the IPG strip is being frozen (at -60 °C or below) for future analysis. Equilibration is always performed
immediately prior to the second-dimension run, never before storage of the Immobiline DryStrip gels.
The second-dimension gel itself should be prepared and ready to accept the Immobiline DryStrip gel
before beginning the equilibration protocol.
3.1.1 Equilibration solution components
The equilibration step saturates the Immobiline DryStrip gel with the SDS buffer system required for the seconddimension separation. The equilibration solution contains buffer, urea, glycerol, reductant, SDS, and dye. An additional
equilibration step replaces the reductant with iodoacetamide.
Equilibration buffer (75 mM Tris-HCl, pH 8.8) maintains the Immobiline DryStrip gel in a pH range appropriate for
electrophoresis.
Urea (6 M) together with glycerol reduces the effects of electroendosmosis by increasing the viscosity of the buffer (3).
Electroendosmosis is due to the presence of fixed charges on the Immobiline DryStrip gel in the electric field and can
interfere with protein transfer from the Immobiline DryStrip gel to the second-dimension gel.
Glycerol (30%) together with urea reduces electroendosmosis and improves transfer of proteins from the first to the
second dimension (3).
Dithiothreitol (DTT) preserves the fully reduced state of denatured, unalkylated proteins.
Sodium dodecyl sulfate (SDS) denatures proteins and forms negatively charged protein-SDS complexes (see section 3.2).
Iodoacetamide alkylates thiol groups on proteins, preventing their reoxidation during electrophoresis. Protein
reoxidation during electrophoresis can result in streaking and other artifacts. Iodoacetamide also alkylates residual
DTT to prevent point streaking and other silver-staining artifacts (80). Iodoacetamide is introduced in a second
equilibration step. The second equilibration with iodoacetamide minimizes unwanted reactions of cysteine residues
(i.e. when mass spectrometry is to be performed on the separated proteins).
Tracking dye (bromophenol blue) allows monitoring of the progress of electrophoresis.
80-6429-60 AD 71
3.1.2 Equilibrating Immobiline DryStrip gels
The second-dimension vertical gel must be ready for use prior to Immobiline DryStrip gel equilibration. If not
using the DryStrip gel immediately, refer to section 2.8.3 for preservation guidelines.
Protocol
Equilibration is carried out in a two-step process using tubes and volume of equilibration solution as specified in Table 22.
Preparatory steps
1. Place the IPG strips in individual tubes, with the support film toward the tube wall.
2. Prepare an appropriate volume of SDS equilibration buffer solution (see appendix I, solution E) then measure into
two equal volumes. Add DTT to one portion (100 mg per 10 ml) and iodoacetamide to the other (250 mg per 10 ml).
Equilibration
1. Add the appropriate volume of SDS equilibration buffer (+ DTT) to each strip. Cap or seal the tubes with flexible
paraffin film and place them on their sides on a rocker for the equilibration process. Equilibrate for 15 min.
2. Pour off buffer from above step and add the appropriate volume of SDS equilibration buffer (+iodoacetamide) to
each strip. Again cap or seal the tubes with flexible paraffin film and place them on their sides on a rocker for the
equilibration process. Equilibrate for an additional 15 min.
Be consistent with the timing of the equilibration steps.
Table 22. Suggested containers and volumes of equilibration solution.
Strip length (cm)
Container
7
Disposable, 15-ml conical tubes
Equilibration solution (ml)
2.5–5
11
25 × 200 mm screw-cap culture tubes
5–10
13
25 × 200 mm screw-cap culture tubes 5–10
18
25 × 200 mm screw-cap culture tubes, Equilibration tubes available from GE Healthcare, or Petri dish
10–15
Equilibration tubes available from GE Healthcare or Petri dish
10–15
24
The subsequent steps of gel assembly, preparation of electrophoresis unit, insertion of the gel into the
precast gel cassette, and melting of the sealing solution can be performed as the Immobiline DryStrip gels are
equilibrating, as long as the timeframes above are adhered to.
3.2 Background to SDS-PAGE
SDS-PAGE is an electrophoretic method for separating polypeptides according to their molecular weights (Mr).
The technique is performed in polyacrylamide gels containing sodium dodecyl sulfate (SDS). The intrinsic electrical
charge of the sample proteins is not a factor in the separation due to the presence of SDS in the sample and the gel.
SDS is an anionic detergent that, when in solution in water, forms globular micelles composed of 70–80 molecules
with the dodecyl hydrocarbon moiety in the core and the sulfate head groups in the hydrophilic shell. SDS and
proteins form complexes with a necklace-like structure composed of protein-decorated micelles connected by short
flexible polypeptide segments (77). The result of the necklace structure is that large amounts of SDS are incorporated
in the SDS-protein complex in a ratio of approximately 1.4 g SDS/g protein. SDS masks the charge of the proteins
themselves and the formed anionic complexes have a roughly constant net negative charge per unit mass. Besides
SDS, a reducing agent such as DTT is also added to break any disulfide bonds present in the proteins. When proteins
are treated with both SDS and a reducing agent, the degree of electrophoretic separation within a polyacrylamide gel
depends largely on the molecular weight of the protein. In fact, there is an approximately linear relationship between
the logarithm of the molecular weight and the relative distance of migration of the SDS-polypeptide complex. (Note:
This linear relationship is only valid for a certain molecular weight range, which is determined by the polyacrylamide
percentage. See Table 24 for the optimum linear separation range for single percentage [homogeneous] and gradient gels.)
72 80-6429-60 AD
The most commonly used buffer system for second-dimension SDS-PAGE is the Tris-glycine system described by
Laemmli (78). This buffer system separates proteins at high pH, which confers the advantage of minimal protein
aggregation and clean separation even at relatively heavy protein loads. The Laemmli buffer system has the
disadvantage of a limited gel shelflife.
Ettan DALT precast gels utilize a buffer system based on piperidinopropionamide (PPA), which combines long shelflife
with the high separation pH of the Laemmli system. Other buffer systems can also be used, particularly the Tristricine system of Schägger and von Jagow (79) for improving resolution of polypeptides with Mr values below 10 000.
ExcelGel precast gels for second-dimension SDS-PAGE on the Multiphor II Electrophoresis System (see chapter 4)
utilize a different Tris-tricine buffer system.
3.3 Electrophoresis using Ettan DALT Large Vertical
electrophoresis systems
Ettan DALTsix and DALTtwelve Large Vertical electrophoresis systems combined with 24-cm-long Immobiline
DryStrip gels offer the highest possible 2-D resolution. Both systems are designed for simplified assembly and rapid
electrophoresis of the second-dimension gel. Ettan DALTsix system accepts up to six large, second-dimension gels (26
× 20 cm) (Fig 2 on page 11). Ettan DALTtwelve system can handle up to 12 large gels (Fig 3 on page 12). When running
fewer gels, unused slots are filled with blank cassette inserts. Safety interlocks prevent the application of power to the
separation unit unless the lid is properly closed. Both units recirculate the buffer so that even gel temperatures are
maintained during electrophoresis.
Most of the steps are common between Ettan DALTsix and Ettan DALTtwelve systems, and thus the protocols presented
below apply to both. Where there are differences (in two instances —preparing the system for electrophoresis and
inserting gels into the system), alternate protocols are presented under the same section number.
Power supply and temperature control unit
The modular Ettan DALTsix system requires an external power supply and thermostatic circulator to control the buffer
temperature. A power supply capable of 100 W constant power output, such as the EPS 601, is recommended for the
fastest separation time. For temperature control, a circulating water bath such as the MultiTemp III should be used.
The operating temperature range of Ettan DALTsix system is 4–40 °C.
Ettan DALTtwelve system is controlled from the Power Supply/Control Unit. The unit supplies a maximum power
output of 200 W with a maximum of 600 V or 1 A. The power supply unit also controls the temperature of the tank
using Peltier elements. The operating temperature range of Ettan DALTtwelve system is 10–50 °C.
Gel caster
Both Ettan DALTsix and Ettan DALTtwelve systems include gel casters to prepare lab-cast gels. Separator sheets
are sandwiched between the gel-casting cassettes for easy removal from the caster following gel polymerization.
Removable front plates allow for simplified loading and removal of the gel cassettes.
DALTsix Gel Caster accommodates either six 1.0-mm or six 1.5-mm gel cassettes with separator sheets. Fewer gels
can be cast by inserting blank cassettes to minimize the volume of casting solution.
DALTtwelve Gel Caster allows fourteen 1-mm-thick gels and thirteen 1.5-mm-thick gels to be cast at one time. Fewer
gels can be cast by inserting blank cassettes to occupy volume not required. The caster has a unique hydrostatic
chamber to add a displacement solution allowing for volume changes of the solution during polymerization and to
produce multiple gels cast to the same height.
Gradient maker
DALTsix Gradient Maker is used in combination with DALTsix Gel Caster for 1-mm-thick and 1.5-mm-thick gels, and
with DALTtwelve Gel Caster for 1-mm-thick gels. It is designed to produce linear gradients of solutions in the volume
range 200–1000 ml. The gradient maker can be used to form convex or concave exponential gradients by the addition of a
one-holed rubber stopper, a piece of rigid tubing, and a piece of flexible tubing. Refer to the Ettan DALTsix user manual
for more information.
80-6429-60 AD 73
DALT gel casting cassettes
DALT gel casting cassettes fit either Ettan DALTsix or Ettan DALTtwelve electrophoresis units. In the standard hinged
cassette, one tall and one short glass plate are hinged together with vinyl spacers glued in place. The simplified design
of the cassette allows for easy assembly, and no clamps are required to seal the cassette.
Standard glass plate sets and low-fluorescent glass plate sets are available for use with Ettan DALT systems. Vinyl
spacers are glued into place as with the standard cassette. The plates are not hinged, which means that a single glass
plate is able to fit into the spot picker when the gel is chemically bound to the plate using Bind-Silane (see appendix V).
DALT Gel 12.5 and DALT Precast Gel Cassette
DALT Gel 12.5 is a precast polyacrylamide gel (25.5 × 19.6 cm, 1 mm thick) for the second dimension of 2-D electrophoresis. The gel is provided already cast onto a plastic support film. The gel is a homogeneous 12.5% polyacrylamide
gel. It is intended to be used in Ettan DALTsix or Ettan DALTtwelve system together with the DALT Buffer Kit. The gel
is formulated for long shelflife and, when used with the buffer kit, generates a discontinuous buffer system yielding
rapid runs with sharp, reproducible results. The gels are inserted into a specially designed reusable cassette and run
vertically in the Ettan DALT systems.
If fluorescent staining/labeling techniques will be used, do not run gels cast on plastic backing, as it can pose
a problem of high background with some dyes during subsequent analysis.
3.3.1 Preparing Ettan DALT system for electrophoresis using precast gels
Protocols for use of Ettan DALTsix and Ettan DALTtwelve differ in two main areas: preparing the system and inserting
the gel into the unit. Thus, where appropriate, separate protocols are provided for the different systems. The first
instance follows.
Protocol: Preparing Ettan DALTsix
For detailed instructions for using Ettan DALTsix system, please refer to the Ettan DALTsix user manual.
Preliminary steps
Place the unit close to a sink for easy rinsing and draining. Connect the tubing leading to and from the heat
exchanger to a thermostatic temperature controller such as MultiTemp III. Do not connect the heat exchanger
to a water tap or any other coolant supply that does not have pressure regulation. Position an EPS 601 Power
Supply conveniently close to the electrophoresis unit.
1. Prepare anode and cathode buffers (stocks included in the DALT Buffer Kit)
Dilute half of the 100× anode (lower) buffer by adding 37.5 ml to 4.5 l of water.
Dilute one bottle of 10× cathode (upper) buffer to 2x buffer to a final volume of 1.2 l with deionized water.
2. Prepare anode assembly
Insert the anode assembly/cassette carrier into the tank. The anode assembly is molded so that it can only be
inserted in one orientation. The side edge of the assembly should fit into the slot in the side of the tank.
3. Fill with anode buffer
Pour the diluted anode buffer into the tank of the Ettan DALTsix Electrophoresis System (Fig 34). Switch on
the pump.
4. Switch on the temperature controller
Switch on the MultiTemp III temperature controller and adjust the temperature to the desired setting.
A temperature of 25 °C is recommended for electrophoresis.
5. Set aside upper chamber
The upper chamber is prepared once the gel has been inserted into Ettan Daltsix. See section 3.3.5.
74 80-6429-60 AD
Fig 34. Filling the Ettan DALTsix electrophoresis unit with anode buffer.
Protocol: Preparing Ettan DALTtwelve
For detailed instructions for using Ettan DALTtwelve system, please refer to the Ettan DALTtwelve user manual.
1. Prepare cathode buffer
Dilute the cathode buffer included in the DALT Buffer Kit to working strength by adding both bottles of 10× cathode
buffer (total volume 250 ml) to 2.25 l distilled or deionized water.
2. Prepare anode buffer
Ensuring that the valve on the separation unit is set to “circulate”, fill the tank to the 7.5 l fill line with distilled or
deionized water. Add the entire contents (75 ml) of the 100× anode buffer included in the DALT Buffer Kit into the tank.
Avoid pouring the 100× anode buffer on the tubing by spreading the tubing slightly with one hand while
pouring the solution with the other (Fig 35).
3. Switch on the separation unit
4. Turn on the pump to mix the buffers and set the separation unit to desired temperature
A temperature of 25 °C is recommended for electrophoresis.
Fig 35. Spreading the tubing elements apart with one hand while pouring the solution with the other (to avoid pouring the 100× anode
buffer onto the tubing).
80-6429-60 AD 75
3.3.2 Inserting DALT Gel 12.5 into DALT Precast Gel Cassette
Protocol
1. Open the gel package
Cut around the package on two sides at approximately 1 cm from the edge to avoid cutting the gel or the support
film. Remove the gel from the package. The gel is cast onto a plastic support film and does not cover the film
entirely. The gel is covered with a protective plastic sheet. Markings on the protective sheet indicate the orientation
of the gel and the direction of electrophoresis. The bottom (+ or anodic) edge of the gel is flush with the edge of the
support film. The support film protrudes approximately 15 mm beyond the top (- or cathodic) edge of the gel and
approximately 5 mm at either side.
2. Open DALT Precast Gel Cassette
Place the gel cassette on the bench top with the hinge down. Apply 1 ml gel buffer onto the glass plate as a streak along
the spacer on the right edge of the glass plate (Fig 36). Add an additional 2 ml of gel buffer to the center of the plate.
Fig 36. Pipetting a streak of gel buffer onto the glass plate. The
arrow indicates the direction of motion in applying the streak.
3. Remove the protective plastic sheet from the gel
Handling the gel only by the side support film margins, hold it (gel-side down) over the glass plate. Ensure that it
is oriented with the cathodic (-) edge of the gel toward the cathodic (-) edge of the cassette. Align the right edge
of the gel with the right edge of the side spacer of the glass plate side, flex the gel downward slightly and lower it
slowly toward the glass plate from right to left. Take care that the bottom (anodic) edge of the gel is flush (within
1 mm) of the bottom (anodic) edge of the glass plate. The protruding side support film margins (but not the gel)
should rest on top of the side spacers.
4. Remove bubbles and excess buffer
Use the roller (a separate accessory) to press out any bubbles or liquid from between the gel and the glass. Press
firmly against the plastic support film with the roller and roll over the entire gel (Fig 37). After rolling, the gel should
adhere firmly to the glass and resist further movement.
5. Close the cassette
Close the cassette, snap the plastic frame to the glass plate (Fig 38) and press the edges tightly together along the
entire side of the cassette. Ensure that the cassette is closed completely; an incompletely closed cassette causes a
strongly curved front.
6. Repeat the procedure for each second-dimension gel to be run
76 80-6429-60 AD
Fig 37. Pressing out air pockets between gel and glass plate.
Fig 38. Closing the DALT Precast Gel Cassette.
3.3.3 Equilibrating Immobiline DryStrip gels
Refer to section 3.1.2. The equilibration procedure is the same whether applying the strip to precast or lab-cast gels.
3.3.4 Applying equilibrated Immobiline DryStrip gels to SDS gels
Both types of DALT gel cassettes (those for precast and for lab-cast gels) have a “longer” glass plate. The cassette
should be laid on the bench with the longer glass plate down, and the protruding edge oriented toward the operator
(Fig 39).
Protocol
1. Position the Immobiline DryStrip gel
Dip the equilibrated Immobiline DryStrip gel (see section 3.1.2) in the SDS electrophoresis buffer (see appendix I,
solution M) to lubricate it. If using the DALT Gel 12.5, the diluted cathode buffer can be used to lubricate the strip.
Place the strip with the acidic end to the left, gel surface up onto the protruding edge of the longer glass plate (Fig 39).
If using a system other than DALTtwelve or DALTsix, position the Immobiline DryStrip gel between the plates on the
surface of the second-dimension gel with the plastic backing against one of the glass plates.
2. Ensure Immobiline DryStrip gel has good contact
With a thin plastic ruler, gently push the Immobiline DryStrip gel down so that the entire lower edge of the Immobiline
DryStrip gel is in contact with the top surface of the slab gel (Fig 40). Ensure that no air bubbles are trapped between
the Immobiline DryStrip gel and the slab gel surface or between the gel backing and the glass plate.
3. Optional: Apply molecular weight marker proteins
Best results are obtained when the molecular weight marker protein solution is mixed with an equal volume of a hot
1% agarose solution prior to application to the IEF sample application piece. The resultant 0.5% agarose will gel and
prevent the marker proteins from diffusing laterally prior to the application of electric current.
Other alternatives are to apply the markers to a paper IEF sample application piece in a volume of 15–20 µl.
For less volume, cut the sample application piece proportionally. Place the IEF application piece on a glass plate and
pipette the marker solution onto it, then pick up the application piece with forceps and apply to the top surface of
the gel next to one end of the Immobiline DryStrip gel. The markers should contain 200–1000 ng of each component
for Coomassie staining and approximately 10–50 ng of each component for silver staining.
4. Seal the Immobiline DryStrip gel in place
The agarose sealing solution prevents the Immobiline DryStrip gel from moving or floating in the electrophoresis buffer.
For precast DALT gels, the agarose blocks the narrow gap(s) between the gel edge(s) and the lateral spacer(s) to
prevent leakage of the upper buffer.
Prepare agarose sealing solution for DALT precast gels using the agarose sealing solution from the DALT Buffer Kit. If
using the Laemmli buffer system, see appendix I, solution N.
Melt each aliquot as needed in a 100 °C heat block (each gel will require 1–1.5 ml). It takes approximately 10 min to
fully melt the agarose.
An ideal time to carry out this step is during Immobiline DryStrip gel equilibration.
80-6429-60 AD 77
Allow the agarose to cool until the tube can be held with your fingers (60 °C) and then slowly pipette the amount required
to seal the Immobiline DryStrip gel in place (Fig 41). Pipette slowly to avoid introducing bubbles. Apply only the minimum
quantity of agarose sealing solution required to cover the Immobiline DryStrip gel. Allow a minimum of 1 min for the
agarose to cool and solidify.
Fig 39. Positioning an equilibrated Immobiline DryStrip
gel on the DALT Precast Gel Cassette.
Fig 40. Pushing the Immobiline
DryStrip gel down to contact the
gel slab.
Fig 41. Sealing the Immobiline
DryStrip gel in place on a DALT precast
gel using agarose sealing solution.
3.3.5 Inserting gels into Ettan DALT electrophoresis units
Two protocols follow, the first for inserting gels into Ettan DALTsix and the second for inserting gels into Ettan
DALTtwelve.
Protocol: Inserting gels into Ettan DALTsix
When the electrophoresis buffer has reached the desired temperature, insert the loaded gel cassettes with the
Immobiline DryStrip gels in place.
1. Insert the cassettes into the cassette carrier (Fig 42) and fill any empty slots with blanks.
2. When all six slots are occupied, adjust the buffer level with distilled water so that the level of the diluted anode
buffer is at the “LBC start fill” line marked on the unit.
3. Seat the upper buffer chamber over the gels (Fig 43).
Lubricate the gasket and cassette with cathodic buffer (e.g. SDS running buffer) to assist in assembly.
4. Fill the UBC with 1.2 l of 2x buffer. (if not already done in the protocol in section 3.3.1).
5. Using a small funnel, quickly fill the narrow space between the upper and lower buffer chambers with anode
buffer or distilled water to the same level as in the upper buffer chamber.
It is important that the anode and cathode buffers are filled to the same height in the Ettan DALTsix buffer
chambers.
6. Attach and close the lid. Connect the power leads to the power supply.
Fig 42. Inserting the cassettes into the cassette carrier.
78 80-6429-60 AD
Fig 43. Seating the upper buffer chamber.
Protocol: Inserting gels into Ettan DALTtwelve
When the electrophoresis buffer has reached the desired temperature, insert the loaded gel cassettes with the
Immobiline DryStrip gels in place.
Gel Cassettes and Blank Cassette Inserts slide much more easily into the unit if they are wet. Distilled or deionized
water from a squirt bottle can be used to wet the cassettes and Blank Cassette Inserts as they are being loaded into
the unit.
1. Load the unit from back to front (Fig 44).
2. Fit Blank Cassette Inserts into any unoccupied slots.
3. When all 12 slots are occupied, the lower buffer level should be slightly below the level of the gaskets. Pour the
diluted (1×) cathode buffer into the upper portion of the tank to the fill line (some of this buffer may drip through
the gasket and mix with the anode buffer during the run, but this will not affect performance or results).
4. Close the lid.
Fig 44. Loading the gel cassettes into Ettan DALTtwelve electrophoresis unit.
80-6429-60 AD 79
3.3.6 Electrophoresis conditions with precast gels for both Ettan DALTsix and
Ettan DALTtwelve
Table 23 lists the recommended conditions for Ettan DALTsix and Ettan DALTtwelve systems. Electrophoresis is
performed at constant power in two steps. Stop electrophoresis when the dye front is approximately 1 mm from the
bottom of the gel.
Temperature control improves gel-to-gel reproducibility, especially if the ambient temperature of the laboratory
fluctuates significantly.
For best results, gels should be run at 25 °C.
After electrophoresis, remove gels from their gel cassettes in preparation for staining or blotting. Precast gels have a
barcode, number, and gel percentage printed on them, which should be noted for orientation.
Table 23. Recommended electrophoresis conditions for second-dimension vertical gels.
Step
Power
(W/gel)
Approximate run
duration (h:min)
Ettan DALTsix (set temperature to 25 °C)
1-mm-thick gels
(lab-cast and precast)
1
2
2
17 (max 100)
0:45
4:00
1.5-mm-thick gels
1
2
5
17 (max 100)
0:30
5:00
Ettan DALTtwelve (set temperature to 25 °C)
1-mm-thick gels
(lab-cast and precast)
1
2
2
17 (max 180)
0:45
4:00
1.5-mm-thick gels
1
2
5
17 (max 180)
0:30
6:00
Overnight runs in Ettan DALTsix (set temperature
to 30 °C, power supply for continuous run)
1.0-mm-thick gels run overnight*
1
16:00
1.5-mm-thick gels run overnight*
1.5
18:30
Overnight runs in Ettan DALTtwelve (set temperature
to 30 °C, power supply for continuous run)
1.0-mm-thick gels run overnight*
1
18:00
1.5-mm-thick gels run overnight*
1.5
17:00
* For the best possible resolution, faster separation times should be used. Use the faster (< 6 h) protocols instead.
3.3.7 Preparing lab-cast gels
Some of the chemicals used in the procedures that follow—acrylamide, N,N’-methylenebisacrylamide,
ammonium persulfate, TEMED, thiourea, DTT, and iodoacetamide—are very hazardous. Acrylamide monomer,
for example, is a neurotoxin and suspected carcinogen. Read the manufacturer’s safety data sheet (MSDS)
detailing the properties and precautions for all chemicals in your laboratory. These safety data sheets should
be reviewed prior to starting the procedures described in this handbook. General handling procedures for
hazardous chemicals include using double latex gloves for all protocols. Hazardous materials should be
weighed in a fume hood while wearing a disposable dust mask. Follow all local safety rules and regulations,
including for disposal.
Quick guide for finding information on gel casting for DALTsix and DALTtwelve
electrophoresis systems
To find gel-casting information quickly, refer to Table 25 for gel volumes required, Table 26 for single percentage gel
recipes, and Table 27 for gradient gel recipes.
The instructions provided below for the preparation of vertical SDS-polyacrylamide gels employ the Tris-glycine
system of Laemmli (78). Vertical second-dimension gels are most conveniently cast several at a time, in a multiple gel
caster (see ordering information). For assembly of the gel cassette, refer to the relevant user manual.
80 80-6429-60 AD
Protocol
1. Select the gel percentage
a. Single percentage gel versus gradient gel. When a gradient gel is used, the overall separation interval is wider and
the linear separation interval is larger. In addition, sharper bands result because the decreasing pore size functions
to minimize diffusion. However, a gradient gel requires more skill to cast. For detailed instructions on gradient
preparation, see the user manual for the relevant electrophoresis unit and multiple gel caster.
Single percentage gels offer better resolution for a particular Mr window. A commonly used second-dimension gel for
2-D electrophoresis is a homogeneous gel containing 12.5% total acrylamide.
Note: Stacking gels are not necessary for vertical 2-D gels.
b. Whether single percentage or gradient, the appropriate percentage gel is selected according to the range of
separation desired (Table 24).
Table 24. Recommended acrylamide concentrations for protein separation.
Acrylamide percentage in resolving gel
Single percentage
Separation size range (Mr × 10-3)
5
36–200
7.5
24–200
10
14–200
12.5
15
Gradient
14–100*
14–60*
5–15
14–200
5–20
10–200
10–20
10–150
* Larger proteins fail to move significantly into the gel.
2. Select gel thickness and calculate casting solution volume
DALT gel casting cassettes with either 1.0- or 1.5-mm-thick spacers can be used. Thinner gels stain and destain more
quickly and generally give less background staining. Thicker gels have a higher protein capacity. Thicker gels are also
less fragile and easier to handle.
Table 25 gives the volumes required for Ettan DALT systems.
Table 25. Volumes required per cast (Ettan DALT systems).
Casting system
Volume (ml)
Ettan DALTsix
6 gels × 1-mm-thick spacers
450
6 gels × 1.5-mm-thick spacers 600
Ettan DALTtwelve
14 gels × 1-mm-thick spacers
900
13 gels × 1.5-mm-thick spacers 1200
3. Calculate the formulation of the gel solution
The recipes given in Table 26 produce 900 ml of solution for a single percentage gel. The recipes in Table 27 produce
450 ml each of light and heavy solution for a gradient gel. These recipes can be scaled up or down, depending on the
volume required.
4. Prepare the gel solution
Make up the gel solution without TEMED or ammonium persulfate.
Note: An optional deaeration step may be performed at this point. To do so, make up the solution in a vacuum flask.
Add a small magnetic stirring bar. Stopper the flask and apply a vacuum for several minutes while stirring on a
magnetic stirrer.
Just before casting the gel, add TEMED and 10% ammonium persulfate. Gently swirl the flask to mix, being careful
not to generate bubbles. Immediately pour the gel.
80-6429-60 AD 81
5. Pour and prepare the gel
Fill the gel cassette to 5–10 mm below the top (no stacking gel layer is required).
Overlay each gel with a layer of water-saturated 1-butanol (1.0 ml) immediately after pouring to minimize gel
exposure to oxygen and to create a flat gel surface.
After allowing a minimum of 2 h for polymerization, remove the overlay and rinse the gel surface with gel storage
solution (see appendix I, solution L).
An alternative to using water-saturated 1-butanol to overlay the gels after casting is to spray the edges
of the cassettes using a 0.1% (w/v) SDS/water solution (using a plant sprayer) such that the edges are covered
by just a few millimeters. This technique helps to avoid curved edges on the gels.
6. Storage of unused gels
Gels not used immediately can be stored for future use at 4 °C for up to two weeks. Gel storage solution (see
appendix I, solution L) is pipetted over the top gel surface and the gel cassette is sealed with flexible paraffin film.
Alternatively, the gel cassettes can be stored fully immersed in gel storage solution.
For further information on the preparation of second-dimension vertical SDS slab gels, refer to the user manuals for
the respective vertical gel unit and multiple gel caster.
Table 26. Single-percentage gel recipes for Ettan DALT systems.*
Final gel concentration
10% 12.5%
15%
Monomer solution (solution G)
300 ml
375 ml
450 ml
4× resolving gel buffer (solution H)
225 ml
225 ml
225 ml
9 ml
9 ml
9 ml
360.7 ml
285.7 ml
210.8 ml
10% SDS (solution J)
Double-distilled water
5 ml
5 ml
5 ml
TEMED†
10% ammonium persulfate† (solution K)
0.30 ml
0.25 ml
0.20 ml
Total volume
900 ml
900 ml
900 ml
* Preparation of stock solutions is described in appendix I, solutions G, H, J, and K. Adjust as necessary for the thickness of the gels
and the number of gels cast.
†
Add after (optional) deaeration.
Table 27. Recipes for gradient gels for Ettan DALT systems.*
Light solution – Final concentration
8% 10%
12%
14%
16%
Monomer solution (solution G)
120 ml
150 ml
180 ml
210 ml
240 ml
4× resolving gel buffer (solution H)
113 ml
113 ml
113 ml
113 ml
113 ml
4.5 ml
4.5 ml
4.5 ml
4.5 ml
4.5 ml
210.5 ml
180.5 ml
150.5 ml
120.5 ml
90.5 ml
10% SDS (solution J)
Double-distilled water
10% ammonium persulfate† (solution K)
TEMED†
Total volume
Heavy solution – Final concentration
1.8 ml
1.8 ml
1.8 ml
1.8 ml
1.8 ml
0.225 ml
0.225 ml
0.225 ml
0.225 ml
0.225 ml
450 ml
450 ml
450 ml
450 ml
450 ml
12% 14%
16%
18%
20%
Monomer solution (solution G)
180 ml
210 ml
240 ml
270 ml
300 ml
4× resolving gel buffer (solution H)
113 ml
113 ml
113 ml
113 ml
113 ml
Glycerol (87% [w/w])
31 ml
31 ml
31 ml
31 ml
31 ml
10% SDS (solution J) 4.5 ml
4.5 ml
4.5 ml
4.5 ml
4.5 ml
119.9 ml
89.9 ml
59.9 ml
29.9 ml
0 ml
1.4 ml
1.4 ml
1.4 ml
1.4 ml
1.4 ml
0.225 ml
0.225 ml
0.225 ml
0.225 ml
0.225 ml
450 ml
450 ml
450 ml
450 ml
450 ml
Double-distilled water
10% ammonium persulfate† (solution K)
TEMED†
Total volume
* Preparation of stock solutions is described in appendix I, solutions G, H, J, and K. Adjust as necessary for the thickness of the gels
and the number of gels cast.
†
Add after (optional) deaeration.
82 80-6429-60 AD
3.3.8 Preparing Ettan DALT electrophoresis units for electrophoresis using
lab-cast gels
For Ettan DALT electrophoresis units, the lower tank requires 1× SDS electrophoresis buffer while the upper chamber
requires buffer of a higher concentration. Prepare the required buffers as described in Table 28 (preparation of stock
solutions is described in appendix I, solutions M and F).
For Ettan DALT Gel 12.5, the DALT Buffer Kit must be used to prepare the anode and cathode buffers described
in section 3.3.1.
Table 28. Tank buffer solutions for Ettan DALT systems with lab-cast Laemmli gels.
System
Anodic buffer Volume
Cathodic buffer
(lower buffer chamber)
(l)
(upper buffer chamber)
Volume
(ml)
Dilute from 10× stock
Ettan DALTsix
1.0-mm gels
1× SDS electrophoresis buffer
~4.3
2× SDS electrophoresis buffer
800
1.5-mm gels
1× SDS electrophoresis buffer
~4.3
3× SDS electrophoresis buffer
800
1× SDS electrophoresis buffer
7.5
2× SDS electrophoresis buffer
2500
Ettan DALTtwelve
1.0- or 1.5-mm gels
Protocol: Preparing Ettan DALTsix for use
1. Fill the electrophoresis tank with 4.3 l of 1× SDS electrophoresis buffer.
2. Turn on the pump.
3. Switch on the MultiTemp III temperature controller and set the desired temperature.
A temperature of 10 °C is recommended for rapid electrophoresis. Equilibrate the buffer temperature to at
least 15 °C before starting the run.
Protocol: Preparing Ettan DALTtwelve for use
Fill the anodic chambers of the tank with 1× SDS electrophoresis buffer.
1. Set the valve on the separation unit to “circulate.” Fill the tank to the 7.5 l fill line with 1× SDS electrophoresis buffer.
2. Switch on the separation unit.
3. Turn on the pump to mix the buffers and set the separation unit to desired temperature.
A temperature of 25 °C is recommended for rapid electrophoresis.
3.3.9 Equilibrating Immobiline DryStrip gels with lab-cast gels
When the buffer tank has reached the desired temperature, start equilibrating the Immobiline DryStrip gel as described in
section 3.1.2. The equilibration procedure is the same whether applying the strip to precast or lab-cast gels.
3.3.10 Applying Immobiline DryStrip gels to lab-cast gels
To apply the Immobiline DryStrip gel to the lab-cast gel, follow the procedure as described in section 3.3.4.
3.3.11 Inserting lab-cast gels into Ettan DALT electrophoresis units
To insert lab-cast gels into Ettan DALT electrophoresis units, follow the procedure described in section 3.3.5.
3.3.12 Electrophoresis conditions with lab-cast gels
Follow the procedure described in section 3.3.6.
3.3.13 Troubleshooting
See section 3.5.
80-6429-60 AD 83
3.4 Electrophoresis using other vertical
electrophoresis systems
Several other electrophoresis units work well for second-dimension separation. Choice is to a large degree dependent
on the length of the Immobiline DryStrip used in the first-dimension.
Two systems, miniVE and SE 260, are ideal for running up to two second-dimension gels with 7-cm Immobiline
DryStrip gels. Spacers (1.0 and 1.5 mm) are available as well as two plate lengths (8 or 10.5 cm).
SE 600 Ruby units can be used to cast and run up to four gels 16 cm in length. Divider plates allow two gels to be
cast and run together on each side of the gel tank. The width of the gels can either be 14 or 16 cm, depending on the
width of the spacers chosen, which allows SE 600 Ruby to accommodate either 11- or 13-cm Immobiline DryStrip
gels, respectively. Several gel casters are available, including a 10-gel caster. Low-fluorescent glass plates are also
available for use in SE 600 Ruby.
SE 600 Ruby requires an external power supply such as the EPS 601 and an external recirculating water bath (such as
MultiTemp III Thermostatic Circulator) if temperature control is desired.
3.4.1 Preparing caster and gel sandwich for miniVE, SE 260,
and SE 600 Ruby electrophoresis systems
Protocol
1. Select gel thickness for the system
Either 1.0- or 1.5-mm-thick spacers can be used for all the smaller vertical formats. Thinner gels stain and destain
more quickly and generally give less background staining. Thicker gels have a higher protein capacity. Thicker gels
are also less fragile and easier to handle.
2. Assemble unit
Mount the clamps, spacers, and glass plates to a sandwich. Put the sandwich into the caster using the cams.
See instructions accompanying unit for full details.
3.4.2 Preparing lab-cast gels for miniVE, SE 260,
and SE 600 Ruby electrophoresis systems
Some of the chemicals used in the procedures—acrylamide, N,N’-methylenebisacrylamide, ammonium
persulfate, TEMED, thiourea, DTT, and iodoacetamide—are very hazardous. Acrylamide monomer, for example,
is a neurotoxin and suspected carcinogen. You should have a manufacturer’s safety data sheet (MSDS)
detailing the properties and precautions for all chemicals in your laboratory. These safety data sheets should
be reviewed prior to starting the procedures described in this handbook. General handling procedures for
hazardous chemicals include using double latex gloves for all protocols. Hazardous materials should be weighed
in a fume hood while wearing a disposable dust mask. Follow all local safety rules and regulations for handling
and disposal of materials.
Quick guide for finding information on gel casting for miniVE, SE 260, and SE 600 Ruby
electrophoresis systems
To find gel-casting information quickly, refer to Table 29 for gel volumes required, Table 30 for single percentage gel
recipes, and Table 31 for gradient gel recipes.
The instructions provided below for the preparation of vertical SDS-polyacrylamide gels employ the Tris-glycine
system of Laemmli (78).
84 80-6429-60 AD
Protocol
1. Select the gel percentage
See section 3.3.7, protocol instruction 1. Select the gel percentage.
2. Calculate the required casting solution volume
The total volume of solution required depends on the gel size, the gel thickness, and the number of gels cast. Table
29 gives volumes of gel solution required per gel.
3. Calculate the formulation of the gel solution
The recipes given in Table 30 produce 100 ml of solution for a single percentage gel. The recipes in Table 31 produce
50 ml each of light and heavy solution for a gradient gel. These recipes can be scaled up or down, depending on the
volume required.
4. Prepare the gel solution
Make up the gel solution without TEMED or ammonium persulfate.
Note: An optional deaeration step may be performed at this point. To do so, make up the solution in a vacuum flask.
Add a small magnetic stirring bar. Stopper the flask and apply a vacuum for several minutes while stirring on a
magnetic stirrer.
Just before casting the gel, add TEMED and 10% ammonium persulfate. Gently swirl the flask to mix, being careful
not to generate bubbles. Immediately pour the gel.
5. Pour and prepare the gel
Fill the gel cassette to 5–10 mm below the top (no stacking gel layer is required).
Overlay each gel with a layer of water-saturated 1-butanol (0.3 ml) immediately after pouring to minimize gel
exposure to oxygen and to create a flat gel surface.
After allowing a minimum of 2 h for polymerization, remove the overlay and rinse the gel surface with gel storage
solution (see appendix I, solution L).
Do not allow the overlay of water-saturated 1-butanol to remain on the gel for more than 2–3 h. If leaving the
gel for a longer period of time, replace the 1-butanol with an overlay of running buffer.
6. Storage of unused gels
Gels not used immediately can be stored at 4 °C for up to two weeks. Gel storage solution (see appendix I, solution L)
is pipetted over the top gel surface and the gel cassette is sealed with flexible paraffin film.
Alternatively, the gel cassettes can be stored fully immersed in gel storage solution.
For further information on the preparation of second-dimension vertical SDS slab gels, refer to the user
manuals for the respective electrophoresis system and multiple gel caster.
Table 29. Volumes required per vertical gel (miniVE, SE 260, and SE 600 Ruby systems).
Casting system
Volume (ml)
miniVE and SE 260 (10 × 10.5 cm plates)
1-mm-thick spacers 10
1.5-mm-thick spacers 15
SE 600 Ruby (18 × 16 cm plates)
2-cm wide × 1-mm thick spacers 30
2-cm wide × 1.5-mm thick spacers 40
1-cm wide × 1-mm thick spacers
30
1-cm wide × 1.5-mm thick spacers 45
80-6429-60 AD 85
Table 30. Single-percentage gel recipes for miniVE, SE 260, and SE 600 Ruby systems.*
Final gel concentration
Monomer solution (solution G)
4× resolving gel buffer (solution H)
10% SDS (solution J)
Double-distilled water
10% ammonium persulfate† (solution K)
TEMED†
Total volume
5% 7.5%
10%
12.5%
15%
16.7 ml
25 ml
33.3 ml
41.7 ml
50 ml
25 ml
25 ml
25 ml
25 ml
25 ml
1 ml
1 ml
1 ml
1 ml
1 ml
56.8 ml
48.5 ml
40.2 ml
31.8 ml
23.5 ml
500 µl
500 µl
500 µl
500 µl
500 µl
33 µl
33 µl
33 µl
33 µl
33 µl
100 ml
100 ml
100 ml
100 ml
100 ml
* Preparation of stock solutions is described in appendix I, solutions G, H, J, and K.
†
Ammonium persulfate and TEMED are added immediately prior to casting the gel.
Table 31. Recipes for gradient gels for miniVE, SE 260, and SE 600 Ruby systems.*
Light solution – Final concentration
Monomer solution (solution G)
4× resolving gel buffer (solution H)
10% SDS (solution J)
Double-distilled water
10% ammonium persulfate† (solution K)
TEMED†
Total volume
Heavy solution – Final concentration
5% 7.5%
10%
12.5%
15%
8.4 ml
12.5 ml
16.5 ml
21.0 ml
25 ml
12.5 ml
12.5 ml
12.5 ml
12.5 ml
12.5 ml
500 µl
500 µl
500 µl
500 µl
500 µl
28.5 ml
24.5 ml
20.0 ml
16.0 ml
12.0 ml
170 µl
170 µl
170 µl
170 µl
170 µl
17 µl
17 µl
17 µl
17 µl
17 µl
50 ml
50 ml
50 ml
50 ml
50 ml
10% 12.5%
15%
17.5%
20%
Monomer solution (solution G)
16.7 ml
21.0 ml
25.0 ml
29.2 ml
33.3 ml
4× resolving gel buffer (solution H)
12.5 ml
12.5 ml
12.5 ml
12.5 ml
12.5 ml
7.5 g
7.5 g
7.5 g
7.5 g
7.5 g
500 µl
500 µl
500 µl
500 µl
500 µl
Sucrose
10% SDS (solution J)
Double-distilled water
16.2 ml
11.7 ml
7.7 ml
3.5 ml
0 ml
10% ammonium persulfate† (solution K)
165 µl
165 µl
165 µl
165 µl
165 µl
TEMED†
16.5 µl
16.5 µl
16.5 µl
16.5 µl
16.5 µl
50 ml
50 ml
50 ml
50 ml
50 ml
Total volume
* Preparation of stock solutions is described in appendix I, solutions G, H, J, and K.
†
Ammonium persulfate and TEMED are added immediately prior to casting the gel.
3.4.3 Preparing miniVE, SE 260, and SE 600 Ruby systems for electrophoresis
For these electrophoresis units, prepare enough 1× SDS electrophoresis buffer according to Table 32 (preparation of
stock solutions is described in appendix I, solution M).
Table 32. Gel tank buffer volumes for miniVE, SE 260, and SE 600 Ruby electrophoresis systems.
System
Gel tank buffer volume (l)
miniVE
1.5
SE 260
0.6
SE 600 Ruby
5
Fill the anode buffer tank with SDS electrophoresis buffer. Set the temperature if applicable.
3.4.4 Equilibrating Immobiline DryStrip gels
To equilibrate the Immobiline DryStrip gel, see section 3.1.2. The equilibration procedure is the same whether applying
the strip to precast or lab-cast gels.
86 80-6429-60 AD
3.4.5 Applying Immobiline DryStrip gels
Protocol
1. Dip the Immobiline DryStrip gel in SDS Buffer.
2. While the SDS gels still are in the gel caster, apply the Immobiline DryStrip gels on top of them. Push the strips
gently down to the gel surface.
3. Seal the Immobiline DryStrip gel in place with melted agarose. See appendix I, solution N, Agarose sealing solution.
3.4.6 Inserting gels into miniVE, SE 260, and SE 600 Ruby systems
Protocol: miniVE
For detailed information, refer to the miniVE user manual.
1. Make sure the sealing plate is in the “half open” position.
2. Lower each module into the tank, seating it in the locating slots.
3. Add the appropriate amount of electrophoresis buffer to the tank and to the upper buffer chamber.
4. Attach or close the lid and connect the power leads to the power supply.
Protocol: SE 260 system
For detailed information, refer to the SE 260 user manual.
1. Apply the SDS gel to the electrophoresis tank.
2. Clamp the gel in position and fill up the anode and cathode buffer chambers.
3. Attach or close the lid and connect the power leads to the power supply.
Protocol: SE 600 Ruby system
For detailed information, refer to the SE 600 Ruby user manual.
1. Fit the slotted gasket in the upper buffer chamber.
2. Put the upper buffer chamber onto the gel sandwiches in the casting stand. Fix the gel sandwich to the upper
buffer chamber with the cams and release the gel sandwich from the caster.
3. Fit the upper buffer chamber with the gel sandwiches onto the lower buffer chamber.
4. Fill the upper buffer chamber with Laemmli SDS buffer.
5. Attach or close the lid and connect the power leads to the power supply.
6. Set the temperature control if desired.
7. Stir the SE 600 Ruby lower tank buffer to maintain an even buffer temperature around the gels.
If using only one gel in SE 600 Ruby and SE 260 units, the second side of the unit will need to be blocked with a bufferdam assembly or two glass plates clamped together (no spacers) to prevent current leakage. For detailed information,
please consult the respective instrument user manuals.
3.4.7 Electrophoresis conditions
Table 33 lists the recommended conditions for miniVE, SE 260, and SE 600 Ruby. Electrophoresis is performed at
constant current in two steps. During the initial migration and stacking period (Step 1), the current is approximately
half of the value required for the separation (Step 2), as can be seen from Table 33.
Stop electrophoresis when the dye front is approximately 1 mm from the bottom of the gel.
For these vertical systems, cooling is optional. However, temperature control improves gel-to-gel reproducibility,
especially if the ambient temperature of the laboratory fluctuates significantly.
For best results, gels should be run at 25 °C.
80-6429-60 AD 87
After electrophoresis, remove gels from their gel cassettes in preparation for staining or blotting. Notch or mark each
gel at the upper corner nearest the “+” or “-” end of the Immobiline DryStrip gel to identify the acidic end of the firstdimension separation.
Table 33. Recommended electrophoresis conditions for second-dimension vertical gels.
Step
Current
(mA/gel)
Approximate run.
duration (h:min)
miniVE and SE 260
1.0-mm-thick gels
1
2
10
20
0:15
1:30*
1.5-mm-thick gels
1
2
15
30
0:15
1:30*
1.0-mm-thick gels
1
2
10
50–20
0:15
2:00–5:00*
1.5-mm-thick gels
1
2
15
60–30
0:15
2:00–5:00*
SE 600 Ruby
* The time shown is approximate. Stop electrophoresis when the dye front is 1 mm from the bottom of the gel.
If running at the higher currents, cooling is highly recommended.
3.5 Troubleshooting
Table 34 lists possible problems that might be encountered during vertical SDS-PAGE and how to solve them.
Table 34. Troubleshooting vertical second-dimension SDS-PAGE.
Symptom Possible cause
Remedy
Insufficient volume of buffer in upper
No current at
or lower reservoir.
start of run
Ensure that both reservoirs contain enough SDS
electrophoresis buffer to contact both upper and
lower electrode wires.
Check for leaks.
Second-dimension
separation proceeds
too slowly
Make fresh solutions.
SDS electrophoresis buffer prepared
incorrectly, or resolving gel buffer
prepared incorrectly.
Current leakage.
Make sure all the slots in the electrophoresis unit
are filled with either a gel or a blank cassette.
Prepare fresh monomer stock solution.
Acrylamide solution is too old.
Gel temperature is not uniform.
Dye front curves up
(smiles) at the edges
Regulate gel temperature using a thermostatic
circulator.
Use the maximum possible volume of buffer in the lower reservoir.
Current or power too high.
Limit current or power to values suggested in Table 23.
Dye front curves
down (frowns)
Gel is poorly polymerized near
the spacers.
Degas the gel solution, or increase the amount of
ammonium persulfate and TEMED by 50%.
Improper instrument assembly
(SE 600 Ruby).
Ensure that the gasket is not pinched.
Leakage of upper reservoir.
continues on following page
88 80-6429-60 AD
Ensure that an adequate level of buffer is in the
upper reservoir.
Table 34. Troubleshooting vertical second-dimension SDS-PAGE (continued).
Symptom Possible cause
Remedy
Second-dimension
separation proceeds
slowly with high current
All of the slots in the sealing assembly are
not occupied by either gel cassettes or
blank cassettes.
Ensure that all slots in the electrophoresis unit
are occupied.
Anodic buffer has mixed with cathodic
buffer from overfilling of either the
cathodic reservoir or the anodic reservoir
(Ettan DALT systems).
Do not pour more than the suggested volume (7.5 l)
into the lower reservoir.
Ensure that the level of the anode buffer does not
come above the sealing assembly when the electrophoresis unit is fully loaded. If excess anode buffer
is in the upper reservoir, it should be removed with
a pipette.
Ensure that the level of cathode buffer does not
come above the air vents in the corners of the upper
reservoir.
Lack of mixing between upper and lower reservoirs can be verified by adding bromophenol blue dye to the lower reservoir prior to loading the unit with gels. Several drops of 1% (w/v) bromophenol blue will impart sufficient color to the anode buffer.
Chemicals.
Incomplete gel polymerization
Use only fresh stocks of the highest-quality reagents.
If the dry ammonium persulfate does not “crackle”
when added to water, replace it with fresh stock.
Increase TEMED or ammonium persulfate concentration, or both.
Oxygen.
Remove oxygen from the gel environment. Degas the monomer solution 5–10 min before pouring and
then overlay the gel surface with water-saturated
1-butanol.
Temperature.
Adjust the gel solution temperature to a minimum of 20 °C, especially for gels with low acrylamide concentration.
Pronounced downward There is an unfilled gap between the
curving of the dye front gel and one of the spacers.
on one or both sides of the DALT Gel 12.5
When sealing the Immobiline DryStrip gel into place on top of the gel, ensure that some of the sealing
solution flows down any gap that may exist between
the gel and spacer.
Ensure cassette(s) are properly closed.
Precast gel cassette(s) not
properly closed.
80-6429-60 AD 89
90 80-6429-60 AD
4. Use of the flatbed Multiphor II Electrophoresis System for first and second dimensions
4.0 Overview
Multiphor II Electrophoresis System is a versatile flatbed system that provides excellent resolution and rapid
separations in large-format gels that are efficiently and uniformly cooled through a ceramic cooling plate connected
to the cooling unit. This improves resolution and speed at high voltages.
The modular design of the Multiphor II Electrophoresis System gives it the flexibility to handle virtually any flatbed
electrophoretic technique. It is particularly well suited for ultra-thin gels (0.1–0.5 mm) on glass or plastic supports up to
sizes of 20 × 26 cm.
Multiphor II Electrophoresis System comprises a buffer tank with four leveling feet, ceramic (aluminum oxide) cooling
plate with accessories, polycarbonate safety lid, and electrode holder with movable EPH/IEF electrodes (for buffer
strips and electrode strips). In addition to accommodating gels of different sizes, the electrodes make secure, uniform
contact with buffer strips, eliminating the need for large volumes of liquid buffers. Buffer strips can be positioned and
held in place using Multiphor II Buffer Strip Positioner.
To complete the Multiphor II Electrophoresis System, EPS 3501 XL Power Supply and MultiTemp III Thermostatic
Circulator are also required.
As described below, Multiphor II Electrophoresis System can be used for both first-dimension IEF and second-dimension
SDS-PAGE. Strip rehydration with or without sample included is performed in the Immobiline DryStrip IPGbox. After
rehydration, the Immobiline DryStrip gels are transferred to the electrophoresis unit for first-dimension IEF using the
Immobiline DryStrip Kit accessory.
4.1 First-dimension IEF using Multiphor II Electrophoresis System and Immobiline DryStrip Kit
Multiphor II Electrophoresis System can be readily configured for first-dimension IEF separations by incorporating an
Immobiline DryStrip Kit, and choosing the Immobiline DryStrip gel and IPG Buffer to match the required pH gradient.
When equipped with Immobiline DryStrip Kit, the system can run up to 12 Immobiline DryStrip gels simultaneously,
with gel lengths up to 24 cm. Focusing time depends on the gel length, pH range, and the nature of the sample but
can be expected to be in the range of 2–72 h.
DryStrip gels are rehydrated using a Reswelling Tray (accessory) in a solution containing the necessary additives and,
optionally, the sample proteins (rehydration solution is described in detail in section 2.6). IEF is performed by gradually
increasing the voltage across the Immobiline DryStrip gels to at least 3500 V, and maintaining this voltage for at least
several thousand Volt-hours. After IEF, the Immobiline DryStrip gels are equilibrated in equilibration solution and
applied onto SDS-polyacrylamide gels for the second-dimension separation.
Sample can be loaded using rehydration loading, cup loading, or paper-bridge loading. Each of these is described in
more detail later in this chapter. Figure 45 provides guidelines for selecting the appropriate mode of sample application.
80-6429-60 AD 91
Multiphor II Electrophoresis System
pH gradient
3.5–4.5
3.0-5.6 NL
4.0-7.0
3.0-7.0 NL
5.3-6.5
3.0-10.0
3.0-10.0 NL
3.0-11.0 NL
6.2-7.5
Analytical
Rehydration
loading
Cup
loading
Preparative
Rehydration paper-bridge
loading
loading
6.0-9.0
6.0-11.0
7.0-11.0 NL
Fig 45. Guidelines for selecting the appropriate mode of sample application in the Multiphor II Electrophoresis System.
For cup loading, sample is pipetted into sample cups precisely positioned on the surface of the Immobiline DryStrip
gels. Up to 100 µl per strip can be applied through sample cups, and up to 850 µl with paper-bridge loading (74).
4.1.1 Immobiline DryStrip gel rehydration—IPGbox
IPGbox allows up to 12 strips (up to 24 cm long) to be rehydrated independently and simultaneously. Samples can be
loaded during rehydration by including them in the rehydration buffer (rehydration loading). Alternatively, samples can
be applied to rehydrated strips via sample cups or paper-bridge loading.
For protocol, see section 2.7 (page 57).
Table 35. Rehydration solution volume per Immobiline DryStrip gel—Multiphor II protocol.
Immobiline DryStrip gel length (cm)
7
Total volume per strip* (µl)
125
11
200
13
250
18
340
24
450
* Including sample, if applied.
Table 36. Troubleshooting Immobiline DryStrip gel rehydration in IPGbox.
Symptom Possible cause
Remedy
Uneven or incomplete
rehydration of strips
Depending on the Immobiline DryStrip gel pH
interval and the pH of the rehydration solution,
either the basic end or the acidic end will
swell faster than the other. The strip may not necessarily be of an even thickness following rehydration.
At the start of rehydration, ensure that the
rehydration solution is evenly distributed
under the Immobiline DryStrip gel.
Move the gel strip back and forth to aid
distribution. The gel strip should float on
the rehydration solution.
Unopened Immobiline DryStrip gel package
was stored at or above room temperature
for too long.
Store Immobiline DryStrip gels sealed at
a temperature below -20 °C.
Immobiline DryStrip gels were stored at or above room temperature for too long.
Do not allow dry Immobiline DryStrip gels
to remain at room temperature for longer
than 10 min as they will pick up moisture
from the air.
Incorrect volume of rehydration solution used.
Make sure the correct amount of rehydration solution according to Table 35 is added to the channel in the Immobiline DryStrip
IPGbox.
Check calibration of pipettors.
Rehydration time is too short.
Rehydrate the Immobiline DryStrip gels for at least 10 h.
92 80-6429-60 AD
4.1.2 Preparing for IEF
The components of the Immobiline DryStrip Kit include a tray and electrode holder, anode and cathode electrodes, an
Immobiline DryStrip aligner, a sample cup bar, and sample cups.
Procedures A and B below should be completed before the Immobiline DryStrip gels are removed from the
Immobiline DryStrip IPGbox.
A. Prepare the Immobiline DryStrip Kit
1. Clean all components of the Immobiline DryStrip Kit
The Immobiline DryStrip tray, Immobiline DryStrip aligner, electrodes, sample cup bar, and sample cups must be
clean and ready for use. Clean with detergent, rinse thoroughly with distilled water, and allow to dry.
2. Confirm electrical connections on Multiphor II Electrophoresis System
Check that the red bridging cable in the Multiphor II unit is connected (seated under the cooling plate).
3. Establish cooling
Set the temperature on MultiTemp III Thermostatic Circulator to 20 °C. Position the cooling plate on the Multiphor II unit
and ensure that the surface is level.
4. Position the Immobiline DryStrip tray
Pipette approximately 3–4 ml of Immobiline DryStrip Cover Fluid onto the cooling plate. Position the Immobiline
DryStrip tray on the cooling plate so the red (anodic) electrode connection of the tray is positioned at the top of the
plate near the cooling tubes. Remove any large bubbles between the tray and the cooling plate; small bubbles can
be ignored. Immobiline DryStrip Cover Fluid serves as an electrical insulating fluid to ensure good thermal contact
between the cooling plate and the tray. Connect the red and black electrode leads on the tray
to the Multiphor II unit.
5. Position the Immobiline DryStrip aligner
Pour approximately 10 ml of Immobiline DryStrip Cover Fluid into the Immobiline DryStrip tray. Place the Immobiline
DryStrip aligner, 12-groove side up, into the tray on top of the Immobiline DryStrip Cover Fluid. The presence of air
bubbles between the strip positions under the aligner will not affect the experiment. For easier visualization of the
grooves in the aligner, avoid getting Immobiline DryStrip Cover Fluid on top of the aligner.
B. Prepare electrode strips
1. Cut electrode strips to size
Cut two IEF electrode strips to lengths of 110 mm each.
2. Soak electrode strips with distilled water
Place the electrode strips on a clean, flat surface such as a glass plate. Soak each electrode strip with
0.5 ml distilled water. Blot with filter paper to remove excess water.
Electrode strips must be damp, not wet. Excess water may cause streaking.
C. IEF with rehydration loading
1. Remove the rehydrated Immobiline DryStrip gel from the Immobiline DryStrip IPGbox
To remove an Immobiline DryStrip gel from the Immobiline DryStrip IPGbox, slide the tip of a pair of forceps along the
sloped end of the channel and into the slight depression under the Immobiline DryStrip gel. Grasp the end of the strip
with the forceps and lift the strip out of the tray.
2. Position the Immobiline DryStrip gel in the Immobiline DryStrip aligner
Immediately transfer the rehydrated Immobiline DryStrip gels (gel side up) to adjacent grooves of the aligner in the
Immobiline DryStrip tray (Fig 48). Place the strips with the acidic ends at the top of the tray near the red electrode
(anode). The other ends should be at the bottom of the tray near the black electrode (cathode).
Align the Immobiline DryStrip gels so the anodic gel edges are lined up.
3. Attach the electrode strips
Place the moistened electrode strips laterally across the cathodic and anodic ends of the aligned Immobiline DryStrip gels.
The electrode strips must at least be in partial contact with the gel surface of each Immobiline DryStrip gel.
80-6429-60 AD 93
4. Position the electrodes
Each electrode has a side marked red (anode) or black (cathode). Align each electrode over an electrode strip,
ensuring the marked side corresponds to the side of the tray giving electrical contact. When the electrodes are
properly aligned, press them down to contact the electrode strips. Check that the Immobiline DryStrip gels are still
aligned in their grooves (Fig 49).
5. Overlay the Immobiline DryStrip gel with Immobiline DryStrip Cover Fluid
Overlay each Immobiline DryStrip gel with 3 ml of Immobiline DryStrip Cover Fluid to minimize evaporation and urea
crystallization.
Fig 48. Positioning Immobiline DryStrip gels in the Immobiline
DryStrip aligner.
Fig 49. Alignment of electrodes over Immobiline DryStrip gels.
4.1.3 Sample application by cup loading
If the sample was not applied by means of the rehydration solution, it can be applied using the sample cups
immediately prior to isoelectric focusing. When sample cups are used, the sample load limits are lower and
more specific.
Guidelines on suitable sample loads for different gradients and Immobiline DryStrip gels are given in Table 16
(see section 2.5). These values should only be regarded as a rough guide. Suitable sample loads will vary greatly
between samples and with the sensitivity of the staining method used.
Protocol
1. Prepare the sample
Prepare the sample in a solution similar in composition to the rehydration solution used.
2. Determine the point of sample application
The optimal application point depends on the characteristics of the sample. When the proteins of interest have
acidic pIs or when SDS has been used in sample preparation, sample application near the cathode is recommended.
Anodic sample application is necessary with pH 6–11 and 6–9 gradients and preferred when pH 3–10 gradients are
used. The optimal application point can vary with the nature of the sample. Empirical determination of the optimal
application point is best.
3. Position the sample cup bar
Place sample cups on the sample cup bar, high enough on the bar to avoid touching the gel surface. Position the
sample cup bar so the sample cups are a few millimeters away from the cathodic or anodic electrode, depending
on your sample. The sample cups must face the electrode. The sample cup bar has a spacer on one side; slide the
sample cup bar toward the anode/cathode until the spacer just touches the anodic/cathodic electrode.
4. Press the sample cups against the Immobiline DryStrip gels
Move the sample cups into position, one sample cup above each Immobiline DryStrip gel, and gently press the
sample cups down to ensure good contact with each Immobiline DryStrip gel (Fig 50). This is the most critical part of
the setup. Check that strips are in their correct, straight position in the Immobiline DryStrip aligner.
94 80-6429-60 AD
5. Apply Immobiline DryStrip Cover Fluid
Once the sample cups are properly positioned, pour 70–80 ml Immobiline DryStrip Cover Fluid into the tray to
completely cover the Immobiline DryStrip gels. If the Immobiline DryStrip Cover Fluid leaks into the sample cups,
remove it with a pipette, correct the leakage, and check for leakage again. Add approximately 150 ml of Immobiline
DryStrip Cover Fluid to completely cover the sample cups. The Immobiline DryStrip gels are submerged under a layer
of Immobiline DryStrip Cover Fluid to prevent drying of the gel, precipitation of the components of the rehydration
solution, and diffusion of gas into the gel.
6. Apply the sample
Apply sample (up to 100 µl per Immobiline DryStrip gel) into the sample cups by pipetting under the surface of the
Immobiline DryStrip Cover Fluid (Fig 51). The sample should sink to the bottom of the cup. Check for leakage.
7. Start IEF
Ensure that the electrodes on the tray are connected and place the lid on the Multiphor II unit. Connect the leads on
the lid to the power supply. Ensure that the current check on the EPS 3501 XL Power Supply is switched off. Begin IEF.
When sample is applied via sample cups, precipitates can form at the application point and the amount
of protein that can be loaded is less than if the sample was included in the rehydration solution. Protein
precipitation and aggregation at the application point can sometimes be avoided by observing the following:
• The sample should contain urea, nonionic detergents, and IPG Buffer or carrier ampholytes.
• Apply the sample in dilute solutions (60–100 µg protein per 100 µl).
For micropreparative applications, rehydration loading is recommended. Paper-bridge loading is recommended if
using basic strips (see section 4.1.4).
Fig 50. Attaching sample cups to the cup bar and pressing them
against Immobiline DryStrip gels.
Fig 51. Applying sample into sample cups.
4.1.4 Paper-bridge loading
Higher sample volumes and protein amounts can be applied with paper bridges that are placed between the anodic
or cathodic end of the Immobiline DryStrip gel and the electrode strip. A large sample volume requires a large paper
pad applied at the opposite end to absorb excess water.
Paper bridges and electrode pads are cut from 1-mm-thick CleanGel electrode strips (see ordering information)
to a size of 15 × 25 mm and with an arrowhead as shown in Figure 52. The rehydrated Immobiline DryStrip gel is
positioned directly on the glass bottom of the Immobiline DryStrip tray. Up to four Immobiline DryStrip gels can be run
simultaneously on the Multiphor II Electrophoresis System. The arrow-headed paper, to which 375 µl sample solution
has been added, is then positioned at the anodic or the cathodic end of the Immobiline DryStrip gel.
To hold the paper bridge and Immobiline DryStrip gel in place, press a sample cup positioned on the sample cup bar down
on top of the arrowhead. A solution containing up to 10 mg protein (in 850 µl sample solution applied to a 15 × 50 paper
bridge) can be loaded on an 18-cm-long, narrow-pH-range Immobiline DryStrip gel under favorable conditions (74). The
application point (anodic or cathodic) is of key importance for obtaining good results.
80-6429-60 AD 95
IPG
strip
Pressure from
sample cup
Paper Electrode
bridge strip
Electrode
Fig 52. Setup for sample application via a paper bridge.
4.1.5 IEF guidelines for Multiphor II Electrophoresis System
IEF using the Multiphor II Electrophoresis System is conducted at high voltages (up to 3500 V) and very low currents
(typically less than 1 mA) due to the low ionic strength within Immobiline DryStrip gels. During IEF, the current
decreases while the voltage increases as proteins and other charged components migrate to their equilibrium
positions. In a typical IEF protocol, voltage is gradually increased to the final desired focusing voltage, which is held for
several hours or more. With cup loading, a low initial voltage minimizes sample aggregation and generally allows the
parallel separation of samples with differing salt concentrations.
The main factors determining the required volt-hours (Vh) are the length of the Immobiline DryStrip gels and the pH
gradient used. Sample composition, rehydration solution composition, and sample application mode influence the
required volt-hours. Table 37 gives volt-hour values suitable for most samples with rehydration loading or anodic cup
loading.
Cathodic sample application on wide-range gradients pH 3–10 requires considerably longer focusing times
than those stated in Table 37, especially if SDS-containing samples are used. As an example, an SDSsolubilized serum protein sample applied at the cathodic end of a pH 3–10 NL gradient requires Volt-hours in
excess of 2–2.5-fold of that stated in Table 37 (75).
Salt and buffer ions in the sample can require an increase of the time for phase 2 compared with the values
given in Table 37, particularly when cup loading is used. High ion concentrations in the sample can also
require an increase of the total Volt-hour requirement, as these ions have to be transported to the ends of the
Immobiline DryStrip gels. Larger quantities of protein require more time to focus.
Focusing for substantially longer than recommended will cause horizontal streaking and loss of proteins. This
phenomenon is called “over-focusing”. Therefore, focusing time should be reduced to the minimum necessary
(see chapter 7, Troubleshooting).
4.1.6 Protocol examples
The protocols in Table 37 are suitable for first-dimension isoelectric focusing of protein samples in typical analytical
quantities (Table 16) with IPG Buffer concentrations of 0.5 to 2% in the rehydration solution. The optimal focusing time
will vary with the nature of the sample, the amount of protein, and how the sample is applied.
For higher protein loads (up to 1 mg or more) the final focusing step of each protocol can be extended by an
additional 20% of the total recommended Volt-hours if necessary.
Sample application onto pH 6–11, pH 6–9, and pH 7–11 NL Immobiline DryStrip gels by rehydration loading is
less likely to give high-quality 2-D results and should be avoided. Samples should be applied using cup loading
at the acidic end of the Immobiline DryStrip gel, coupled with use of DeStreak Reagent.
96 80-6429-60 AD
4.1.7 Running a Multiphor II protocol
Ensure that the electrodes in the Immobiline DryStrip tray are connected and place the lid on the Multiphor II unit.
Connect the leads on the lid to the power supply. Ensure that the current check on the EPS 3501 XL Power Supply is
switched off. Begin IEF.
As isoelectric focusing proceeds, the bromophenol blue tracking dye migrates toward the anode. Note that
the dye front leaves the Immobiline DryStrip gel well before focusing is complete, so clearing of the dye is no
indication that the sample is focused. If the dye does not migrate, no current is flowing. If this occurs, check
the contact between the electrodes and the electrode strips. Also check that the electrode leads and bridging
cable are correctly connected, and check that the electrodes are positioned properly so that the marked side
contacts the side rail.
The protocols below are suitable for running 7–24-cm Immobiline DryStrip gels on the Multiphor II Electrophoresis
System connected to EPS 3501 XL Power Supply.
The focusing times given in Table 37 are guidelines only. They may vary with the nature of the sample and how
the sample is applied. If using crude samples with high protein and salt content or using paper-bridge loading,
the run time in total kilovolt-hours should be increased by 10%.
Table 37. Guidelines for running Immobiline DryStrip gels on Multiphor II Electrophoresis System. Running conditions: Temperature 20°C;
current 2 mA total; power 5 W total. Program EPS 3501 XL Power Supply in gradient mode and with current check option turned off.
7-cm strips
pH intervals Step 3–11 NL 3–10 6–11 Voltage (V) Time (h) kVh
1
200 2
3500 3
3500 Total 0:01
1:30 0:40–1:05 2:10–2:35 2.8
2.2–3.7
5.0–6.5
3–10 NL 4–7 3–5.6 NL 1
200 2
3500 3
3500 Total 0:01
1:30 0:55–1:30 2:25–3:00 2.8
3.2–5.2
6.0–8.0
7–11 NL 1
300 2
3500 3
3500 Total 0:01
1:30 1:10–2:02 2:40–3:30 2.9
4.1–7.1
7.0–10.0
5.3–6.5 6.2–7.5 1
300 2
3500 3
3500 Total 0:01
1:30 2:36–3:45 4:06–5:15 2.9
9.1–13.1
12.0–16.0
pH intervals Step Time (h) kVh
3–11 NL 3–10 6–11 1
300 2
3500 3
3500 Total 0:01
1:30 1:45–2:35 3:15–4:05 2.9
6.1–9.1
9.0–12.0
4–7 3–5.6 NL
1
300 2
3500 3
3500 Total 0:01
1:30 2:20–3:30 3:50–5:00 2.9
8.1–12.1
11.0–15.0
7–11 NL 1
300 2
3500 3
3500 Total 0:01
1:30 3:30–4:55 5:00–6:25 2.9
12.1–17.1
15.0–20.0
5.3–6.5 6.2–7.5 1* 500* 2
3500 3
3500 Total 0:01*
1:30 7:10–9:10 8:40–10:40* 3.0
25.0–32.0
28.0–35.0
11-cm strips
Voltage (V) * To adjust this protocol for an overnight run, extend step 1 by 5 h (2.5 kVh) and reduce step 3 by 2.5 kVh.
80-6429-60 AD 97
13-cm strips
pH intervals Step Time (h) kVh
3–10 3–11 NL 6–11 1
300 2
3500 3
3500 Total Voltage (V) 0:01
1:30 3:10–4:00 4:40–5:30 2.9
11.1–14.1
14.0–17.0
3–10 NL 4–7 3–5.6 NL 1
300 2
3500 3
3500 Total 0:01
1:30 3:45–5:10 5:15–6:40 2.9
13.1–18.1
16.0–21.0
7–11 NL 1
500 2
3500 3
3500 Total 0:01
1:30 5:10–6:20 6:40–7:50 3.0
18.1–22.0
21.0–25.0
5.3–6.5
6.2–7.5 1* 500* 2
3500 3
3500 Total 0:01*
1:30 10:00–12:50 11:30–14:20 3.0
35.0–45.0
38.0–48.0
* To adjust this protocol for an overnight run, extend the time of step 1 to 2h.
18-cm strips
pH intervals Step Time (h) kVh
3–10 3–11 NL 6–11 1
500 2
3500 3
3500 Total Voltage (V) 0:01
1:30 4:50–6:20 6:20–7:50 3.0
17.0–22.0
20.0–25.0
4–7 3–10 NL 3–5.6 NL 1
500 2* 500 3
3500 4
3500 Total 0:01
6:00 1:30 5:25–9:25 12:55–16:55 3.0
3.0
19.0–30.0
25.0–36.0
6–9 7–11 NL 1
500 2* 500 3
3500 4
3500 Total 0:01
3:00 1:30 10:10–13:00 14:40–17:30 1.5
3.0
35.5–45.5
40.0–50.0
5.3–6.5 6.2–7.5 1
500 2
3500 3
3500 Total 0:01
1:30 19:10–23:25 20:40–24:55 3.0
67.0–82.0
70.0–85.0
* This step is added to give a convenient overnight run (15 h). This step may be omitted. Step 4 should then be extended by 2.5 kVh.
98 80-6429-60 AD
24-cm strips
pH intervals Step Duration (h:min) kVh
3–11 NL 3–10 1
500 2* 500* 3
3500 4
3500 Total Voltage (V) 0:01 5:00* 1:30 8:30-11:20 15:00–17:50 2.5
3.0
29.5-39.5
35.0–45.0
3–10 NL 4–7
3–7 NL 3–5.6 NL
1
500 2
3500 3
3500 Total 0:01 1:30 12:00–16:20 13:30–17:50 3.0
42.0–57.0
45.0–60.0
6–9 7–11 NL 1
500 2
3500 3
3500 Total 0:01 1:30 16:20–22:00 17:50–23:30 3.0
57.0–77.0
60.0–80.0
3.5–4.5 1
500 2
3500 3
3500 Total 0:01 1:30 22:00–27:40 23.30–29:10 3.0
77.0–97.0
80.0–100.0
5.3–6.5 6.2–7.5 1
500 2
3500 3
3500 Total 0:01 1:30 30:35–36:20 32.06–37:50 3.0
107.0–127.0
110.0–130.0
* This step is added to give a convenient overnight run (15 h). This step can be omitted. Step 4 should then be extended by 2.5 kVh.
4.1.8 Preservation of focused Immobiline DryStrip gels
After IEF is complete, proceed to the second-dimension separation immediately or store the Immobiline DryStrip gels
at -60 °C or below. This can be conveniently done by placing the strips between plastic sheets, as suggested by Görg
et al. (3) or on glass plates covered in plastic wrap. Alternatively, the DryStrip gels can be stored in screw-cap tubes.
The 7-cm strips fit in disposable 15-ml conical tubes; 11-, 13-, and 18-cm strips fit in 25 × 200 mm screw cap culture
tubes; and 18- and 24-cm strips fit in Equilibration Tubes (see ordering information). The equilibration process is
discussed in section 3.1.
80-6429-60 AD 99
4.1.9 Troubleshooting
Table 38 lists possible problems that could be encountered during IEF using Multiphor II Electrophoresis
System and how to solve them.
Table 38. Troubleshooting first-dimension IEF: Multiphor II Electrophoresis System and Immobiline DryStrip Kit.
Symptom Possible cause
Remedy
Sample cups leak
Incorrect handling and
placement of sample cups.
Sample cups are fragile and should not be used too many times.
Make sure the sample cups are aligned with the Immobiline DryStrip gels. Make sure the bottoms of the sample cups are flat against the surface of the Immobiline DryStrip gels.
Note: Leaks can often be detected prior to sample application:
• Observe the Immobiline DryStrip Cover Fluid when it is poured into the Immobiline DryStrip Kit tray. If it leaks in through the bottom of the sample cups, reposition the cups, remove the cover fluid with a pipette, and check for leakage again.
• An optional check for leakage is to add 0.01% bromophenol blue dye solution to the cups. If the dye leaks out of a cup, it must be corrected. (Important: the leaked detection dye must be removed from the sample cup before loading the sample.)
Low current
This is normal for Immobiline
DryStrip gels, which have
very low conductivity.
An Immobiline DryStrip gel run usually starts at 50–100 µA/strip and drops during the run to below 10 µA/strip.
EPS 3501 XL Power Supply
cannot detect the low µA
range current and shuts off.
Because the EPS 3501 XL Power Supply can operate under very
low currents, it is recommended for use with Immobiline DryStrip
Kit and Immobiline DryStrip gels. Make sure the low-current shut-
off has been bypassed (see 3501 XL Power Supply instructions).
IPG runs may start in a current range that is not detectable by
the EPS 3501 XL Power Supply.
Always include IPG Buffer or Pharmalyte in the rehydration solution.
IPG Buffer omitted from
rehydration solution.
No electrode contact or lack
No current at
start of run
of electrical continuity.
Check that all Multiphor II contacts are in place.
Make sure the metal band within the electrode contacts the metal band along the side of the Immobiline DryStrip tray.
Note that the metal band within the electrode is only on the end
marked with the red or black circle. Ensure that the bridging cable under the cooling plate is properly installed.
Ensure that the Immobiline DryStrip gels are rehydrated along
its entire length.
Immobiline DryStrip gels
are improperly rehydrated.
The high-voltage lead from the electrophoresis unit is supply correctly.
Ensure that the plugs on the high-voltage leads fit securely into the output jacks on the power supply. Use the appropriate not plugged into the power
adapter if necessary.
Sample dye does
not move out of
the sample cup
It is normal for several hours
to elapse before the sample
dye leaves the sample cups.
The sample cups were pressed down so hard against the gel that
they pushed through the gel to
rest against the plastic backing.
This blocks the current and
physically prevents the protein
from entering the Immobiline
DryStrip gels.
The ionic strength of the sample Dilute the sample as much as possible or, just prior to loading,
is higher than the gel. As a result, dialyze the sample to remove salts.
the field strength in the sample
zone is inadequate to move the
protein out of the sample zone at
an appreciable rate.
Sparking or burning
Conductivity of the
of Immobiline
sample/Immobiline
DryStrip gel is too high.
DryStrip gels
100 80-6429-60 AD
Replace Immobiline DryStrip gel and re-apply sample cup.
Ensure the sample is adequately desalted.
Alternatively, before raising the voltage to maximum, include a
prolonged low-voltage phase in the IEF protocol to allow the ions to move to the ends of the Immobiline DryStrip gel.
4.2 Second-Dimension SDS-PAGE using Multiphor II
Electrophoresis System
As discussed in chapter 2, after IEF it is important to proceed immediately to gel equilibration, unless
the IPG strip is being frozen (at -60 °C or below) for future analysis. Equilibration is always performed
immediately prior to the second-dimension run, never prior to storage of the Immobiline DryStrip gels.
See section 4.1.8 for details on preservation of the gels.
The second-dimension gel itself should be prepared and ready to accept the Immobiline DryStrip gel prior to
equilibration.
Before proceeding further, refer to sections 3.1.1 and 3.1.2 for a discussion of the equilibration process.
Note especially the discussion referring to the equilibration solution components and the need for a second
equilibration step with iodoacetamide.
4.2.1 ExcelGel preparation
Two sizes of precast ExcelGel SDS gels are recommended for 2-D electrophoresis:
• ExcelGel SDS 2-D Homogeneous 12.5 (11 × 25 cm)
• ExcelGel SDS Gradient XL 12–14 (18 × 25 cm)
Both gels accept a single 24-, 18-, or 13-cm Immobiline DryStrip gel, two 11-cm, or three 7-cm Immobiline DryStrip
gels. Placing shorter Immobiline DryStrip gels end-to-end is ideal for comparative studies. For maximum resolution,
the larger gel coupled with the 24-cm or 18-cm Immobiline DryStrip gel is the best choice. Using the buffer strip
positioner helps to get optimal results; good reproducibility is achieved because of standardized placement of
Immobiline DryStrip gels and buffer strips, and a straight run because the gel surface is covered.
A flatbed second-dimension system is not recommended if the first dimension has been run on a pH 6–9,
6–11, or 7–11 NL Immobiline DryStrip gel.
Protocol
1. Equilibrate the Immobiline DryStrip gels
During the preparation of the ExcelGel SDS gel, equilibrate the Immobiline DryStrip gels as described in section 3.1.2.
2. Prepare the Multiphor II Electrophoresis System
Set the temperature on the MultiTemp III Thermostatic Circulator to 15 °C. Pipette 2.5–3.0 ml of Immobiline DryStrip
Cover Fluid onto the Multiphor II cooling plate.
3. Position the ExcelGel SDS gel
Remove the gel from the foil package by cutting away the edges of the package. A notch at the lower left-hand
corner of the film identifies the anodic side.
Note: The gel is cast onto a plastic support film and does not cover the film entirely. Both gel types contain a stacking
gel zone with 5% acrylamide. Markings on the plastic cover indicate the direction of electrophoresis. Orient the gel
according to these markings, remove the cover, and place the gel on the cooling plate. The cathodic edge of the
ExcelGel SDS must align and make uniform contact with the cathodic edge of the grid on the cooling plate.
Avoid trapping bubbles between the gel and the cooling plate. Avoid getting Immobiline DryStrip Cover Fluid
on the gel surface as this may cause the buffer strips to slide during electrophoresis.
Separation quality is improved if the gel surface is allowed to dry, uncovered, for approximately 5 min before
proceeding.
4. Place the Multiphor II Buffer Strip Positioner
The pegs protruding from the bottom of the positioner should be in contact with the shorter sides of the cooling
plate. Match the cathode (-) and anode (+) symbols on the positioner to the cathode and anode symbols on the
cooling plate. Slide the positioner so that the cathodic (-) edge of the gel bisects the slot at position 1 (see instructions
provided with Multiphor II Buffer Strip Positioner). Lock the positioner in place by turning the gray locking cam until
the positioner cannot be moved.
80-6429-60 AD 101
5. Position the cathodic buffer strip
Carefully peel back the foil on the colorless cathodic (-) ExcelGel SDS buffer strip. Place the buffer strip with the
smooth, narrow face downward. Align the buffer strip with the edge of the slot at position 1 and place it in the
slot (Fig 53). If the buffer strip breaks, piece it together on the gel.
Vinyl gloves tend to stick less to the buffer strips than other types of plastic gloves. If sticking persists, dampen the
gloves with distilled water or a 5% SDS solution.
6. Position the anodic buffer strip
Carefully peel back the foil on the yellow-colored (+) anodic strip and place it in the appropriate slot of the positioner:
For 11 × 25 cm ExcelGel SDS gels, place the anodic strip in slot 3, in the center of the positioner.
For 18 × 25 cm ExcelGel SDS gels, place the anodic strip in slot 4, anodic edge (+) of the positioner.
The buffer strips should sit snugly within the slots.
4.2.2 Applying equilibrated Immobiline DryStrip gels
Protocol
1. Drain moisture from Immobiline DryStrip gels (flatbed second-dimension only)
After equilibration, place the Immobiline DryStrip gels on filter paper moistened with deionized water. To help
drain the equilibration solution, place the Immobiline DryStrip gels so they rest on an edge. They can be left in this
position for up to 10 min without noticeably affecting the spot sharpness. Alternatively, the Immobiline DryStrip
gels can be gently blotted with moistened filter paper to remove excess equilibration buffer.
2. Position the Immobiline DryStrip gel(s)
Once the equilibrated Immobiline DryStrip gels have drained for at least 3 min, use forceps to place them gel-side
down on the ExcelGel through the slot at position 2 (Fig 54). The anodic side of the IPG DryStrip should be oriented
such that it is toward the front edge of the gel.
Fig 53. Positioning the anodic buffer strip on Multiphor II unit.
Fig 54. Positioning equilibrated Immobiline DryStrip gels on
Multiphor II unit such that the anodic (acidic) side of the strip
is toward the front edge of the gel.
3. Position sample application pieces
Using forceps place one IEF sample application piece at the end of each Immobiline DryStrip gel underneath the
plastic “tab” formed by the overhanging gel support film at each end of the Immobiline DryStrip gel. Be sure the
application pieces touch the ends of the Immobiline DryStrip gel (Fig 55).
Note: Application pieces absorb water that flows out of the Immobiline DryStrip gels during electrophoresis.
4. Ensure contact between Immobiline DryStrip gel and ExcelGel
Make sure that the Immobiline DryStrip gel is in full, direct contact with the SDS gel. To remove any bubbles, stroke
the plastic backing of the Immobiline DryStrip gel gently with a spatula or forceps.
102 80-6429-60 AD
5. Optional: Apply molecular weight marker proteins
If loading marker proteins, place an extra application piece on the surface of the gel just beyond the end of the
Immobiline DryStrip gel. Pipette the markers onto the extra sample application piece. Apply the markers in a volume
of 15–20 µl. For less volume, cut the sample application piece proportionally. The markers should contain 200–1000
ng of each component for Coomassie staining and approximately 10–50 ng of each component for silver staining.
6. Position electrodes
Place the IEF electrode holder on the electrophoresis unit, in the upper position, and align the electrodes with the
center of the buffer strips. Plug in the electrode connectors and carefully lower the electrode holder onto the buffer
strips (Fig 56).
Fig 55. Positioning application pieces.
Fig 56. Positioning electrodes.
4.2.3 Electrophoresis conditions
Place the safety lid on the Multiphor II unit. Connect the power supply. Recommended electrical settings and running
times are listed in Table 39.
Table 39. Electrophoresis conditions for ExcelGel gels.
ExcelGel SDS Homogeneous 12.5
Step
Voltage (V)
Current (mA)
Power (W)
Duration (h:min)
1
120
20
30
0:40
Open the lid and carefully remove the electrodes*
2
600
50
30
1:10†
ExcelGel Gradient XL 12–14
1
200
20
20
0:40
Open the lid and carefully remove the electrodes*
2
800
40
40
2:40†
* Remove the Immobiline DryStrip gel and the application pieces. Then move the cathodic buffer strip forward to cover the area of the
removed Immobiline DryStrip gel. Adjust the position of the cathodic electrode.
†
Stop electrophoresis 5 min after the bromophenol blue front has reached the anodic buffer strip. Remove and discard the buffer strips.
80-6429-60 AD 103
4.2.4 Troubleshooting
Table 40 lists possible problems that could be encountered during second-dimension SDS-PAGE using the
Multiphor II Electrophoresis System and how to solve them.
Table 40. Troubleshooting second-dimension SDS-PAGE: Multiphor II Electrophoresis System.
Symptom Possible cause
Remedy
No current at start of run
The electrode cable is not plugged in.
Ensure that all cables are properly connected.
Dye front curves up
Cathodic buffer strip not in contact
(smiles) at one edge
with the gel at one edge.
Ensure that the cathodic buffer strip is centered
and covers the entire width of the seconddimension gel.
Inadequate cooling.
Dye front curves up
(smiles) at both edges
Ensure that the thermostatic circulator is connected
to the Multiphor II unit and functioning correctly.
Dye front is irregular
Ensure that the expiration dates on the buffer
strips and ExcelGel gels have not elapsed.
Some dye front irregularity results from the use of IPG Buffer and does not affect results.
Buffer strips or ExcelGel gels are old.
Bubbles under the buffer strip.
Ensure that the buffer strips are placed firmly on
the gel with no air bubbles trapped beneath them.
Bubbles under the Immobiline
DryStrip gel.
Ensure that the Immobiline DryStrip gel is placed firmly on the gel with no air bubbles trapped
underneath. Stroke the plastic backing of the
Immobiline DryStrip gel gently with a pair of forceps
to remove trapped bubbles.
Incorrect electrode placement.
Buffer strip slides out
from under the electrode
Ensure that the electrodes are aligned over the
center of the buffer strips before lowering the
electrode holder.
104 80-6429-60 AD
5. Visualizing and evaluating results
5.0 Visualizing results—labeling and staining
Most detection methods used for SDS gels can be applied to second-dimension gels.
The following features are desirable:
• High sensitivity
• Wide linear range for quantitation
• Compatibility with mass spectrometry
• Low toxicity and environmentally friendly
However, because none of the existing techniques can meet all these requirements, a 2-D electrophoresis laboratory
may need to have more than one of the following methods in its repertoire:
Autoradiography and fluorography are the most sensitive detection methods (down to 200 fg of protein). To
employ these techniques, the sample must contain protein radiolabeled in vivo using either 35S, 14C, 3H or, in the
case of phosphoproteins, 32P or 33P. For autoradiographic detection, the gel is simply dried and exposed to X-ray film
or—for quicker results and superior dynamic range of quantitation—to a storage phosphor screen. Fluorography is
a technique that provides extra sensitivity by impregnating the gel in a scintillant such as PPO (2,4-diphenyloxazole)
prior to drying.
Silver staining is a sensitive non-radioactive method (below 1 ng). Silver staining is a complex, multi-step process
utilizing numerous reagents for which quality is critical. It is therefore often advantageous to purchase these reagents
in the form of a dedicated kit, in which the reagents are quality ensured specifically for the silver-staining application.
PlusOne Silver Staining Kit, Protein combines high sensitivity with ease of use.
By omitting glutardialdehyde from the sensitizer and formaldehyde from the silver nitrate solution, the method
becomes compatible with mass spectrometry analysis (81), although at the expense of sensitivity.
When staining DALT precast gels with PlusOne Silver Staining Kit, Protein, a modified staining protocol should be used.
For details of the modified protocol, see appendix II—Optimized silver staining of DALT precast gels using PlusOne
Silver Staining Kit, Protein.
Coomassie staining, although 50- to 100-fold less sensitive than silver staining, is a relatively simple method and
more quantitative than silver staining. Coomassie blue is preferable when relative amounts of protein are to be
determined by densitometry. Colloidal staining methods are recommended, because they show the highest sensitivity,
down to 100 ng/protein spot (82,83). See also appendix III.
Negative Zinc-Imidazole staining has a detection limit of approximately 15 ng protein/spot (85) and is compatible
with mass spectrometry, but is a poor quantitation technique.
Fluorescent labeling (5) and fluorescent staining (86) provide significant advantages over Coomassie blue or
silver staining. Fluorescent detection offers increased sensitivity, simple, robust staining protocols, and quantitative
reproducibility over a broad dynamic range. The method is also compatible with mass spectrometry.
Deep Purple™ Total Protein Stain
Deep Purple Total Protein Stain from GE Healthcare is a fluorescent stain that provides:
• High sensitivity
• Clear, easily discernible, and accurately quantitated protein spots and bands
• High signal-to-noise ratios so low-intensity spots and bands are detected
• Compatibility with most fluorescent scanners and CCD cameras, UV transilluminators, and some light boxes
• Ease of disposal and environmental friendliness (naturally occurring fluorophore is free from heavy metals)
• Low viscosity and thus easy to handle with no oily residue
80-6429-60 AD 105
Deep Purple Total Protein Stain is compatible with downstream analysis such as MS and Edman sequencing and is
ideal for post-staining gels used in 2-D DIGE analysis with Ettan DIGE system (see chapter 6).
Alternatives to Deep Purple Total Protein Stain include Sypro dyes (87–90), which have a sensitivity between colloidal
Coomassie and modified staining using PlusOne Silver Staining Kit, Protein (84). Deep Purple Total Protein Stain
provides superior 1-D and 2-D gel image data compared with Sypro Ruby dyes, and clearer backgrounds (see
additional reading and reference material).
Refer to appendix IV for the protocol for use of Deep Purple Total Protein Stain.
Figure 57 shows 2-D gels stained with Sypro Ruby and Deep Purple Total Protein Stain.
A)
B)
Fig 57. 2-D gels of a protein sample consisting of a mix of HBL100 breast cell line and BT474 breast cell carcinoma stained with (A)
Sypro Ruby and (B) Deep Purple Total Protein Stain. For clarity, the gel images show pH 3–8 where most of the proteins are present. The
expanded region of the gel stained with Sypro Ruby (gel A) and resulting 3-D plot demonstrate the drawbacks associated with “speckling”.
Staining with Deep Purple Total Protein Stain (gel B) eliminates speckling and improves spot clarity, which allows more accurate spot
detection and protein identification. First dimension: pH 3–10 NL 24-cm Immobiline DryStrip strip run on Ettan IPGphor 3 IEF System;
second dimension: 12.5% SDS electrophoresis gel run on Ettan DALTtwelve electrophoresis system. Scanned using Typhoon 9410
Variable Mode Imager. Full experimental details can be found in application note 18-1177-44.
The plastic backing on precast gels can pose a problem of high background when fluorescent staining and
labeling techniques are used.
5.0.1 Automating processing and preserving the gel
Processor Plus automates multistep staining processes for increased convenience and reproducibility. Automated
protocols have been developed to use PlusOne Silver Staining Kit, Protein to silver stain proteins in SDS gels. This
convenient adaptation gives reproducible results and sensitivity below 1 ng per spot for most proteins. With a
modification for subsequent mass spectrometry, detection down to approximately 5 ng per spot can be achieved
(84). For further information regarding methodology, please refer to the Processor Plus Protocol Guide (see additional
reading and reference material).
Staining Tray Set provides a convenient means of staining up to four large-format gels at a time—film-backed, as well
as unbacked. The set includes two stainless steel trays and a perforated stainless steel tray, which seats within the
staining trays, and a transparent plastic cover. The perforated insert supports and restrains gels for transfer between
staining trays while allowing staining solution to drain rapidly.
106 80-6429-60 AD
The film-supported DALT and ExcelGel precast gels are optimally stored in sheet protectors after soaking them in
10% v/v glycerol for 30 min. Unbacked gels are shrunk back to their original sizes by soaking them in 30% (v/v)
methanol or ethanol/4% glycerol until they match their original sizes. For autoradiography the gels are dried onto
strong filter paper with a vacuum dryer.
5.1 Blotting
Second-dimension gels can be blotted onto a nitrocellulose or polyvinylidene difluoride (PVDF) membrane for
immunochemical detection of specific proteins or for chemical microsequencing.
GE Healthcare offers a range of blotting membranes and equipment for such purposes. Hybond™-ECL™ is an
unsupported, 100% pure nitrocellulose membrane that has been validated for use with ECL Western Blotting System
and for all radioactive, non-radioactive, and chromogenic protein blotting applications. It has excellent sensitivity,
resolution, and low background. Hybond-P is a PVDF membrane optimized for use in protein transfers. It has higher
mechanical strength than unsupported nitrocellulose and a protein binding capacity of 125 µg/cm2. Hybond-P is
chemically stable, allowing the use of a range of solvents for rapid de-staining.
The plastic backing on DALT and ExcelGel precast gels is removed with the Film Remover prior to
electrotransfer (see ordering information).
5.2 Evaluating results
In theory, the analysis of up to 15 000 proteins should be possible in one gel; in practice, however, 5000 detected
protein spots means a very good separation. Evaluating high-resolution 2-D gels by a manual comparison of two gels
is not always possible. In large studies with patterns containing several thousand spots, it may be almost impossible
to detect the appearance of a few new spots or the disappearance of single spots. Image collection hardware and
image evaluation software are necessary to detect these differences as well as to obtain maximum information from
the gel patterns.
ImageMaster™ 2D Platinum and DeCyder™ 2-D Differential Analysis software, together with ImageScanner™
and/or Typhoon multicolor fluorescence and phosphor image scanner, comprise a system that allows the user to
capture, store, evaluate, and present information contained in 2-D gels:
• ImageScanner II desktop instrument captures optical information in the visible wavelength range over a range from
0 to more than 3.4 O.D. in reflection or transmission mode. It scans 20 × 20 cm in 40 s at 300 dpi.
• Typhoon 9400 Variable Mode Imager has red-, green-, and blue-excitation wavelengths and a wide choice of
emission filters that enable imaging of a variety of fluorphores.
Typhoon series imagers can be used for high-performance four-color automated fluorescence detection making them
ideal for use with the three-dye system employed in 2-D DIGE analysis with Ettan DIGE system. In addition, Typhoon
9400 series imagers perform storage phosphor imaging and chemiluminescence imaging. Comprehensive information
on fluorescence imaging can be found in the GE Healthcare handbook: Fluorescence imaging, principles, and methods
(see additional reading and reference material).
• ImageMaster 2D Platinum is a high-throughput 2-D imaging software for almost parameter-free spot detection. No
manual spot editing is required, resulting in maximum reproducibility of evaluation results. Matching is based on
spot features rather than simply spot positions. A wide selection of statistical tools enables the user to extract the
relevant information in a minimum of time, with maximum confidence.
• DeCyder 2-D Differential Analysis Software has been specifically developed as a key element of the Ettan DIGE
system and is described further in chapter 6.
In addition to these products, Personal Densitometer™ SI is also available. Personal Densitometer is a highly sensitive,
laser-based transmission densitometer with a linear range of 0.1–3.5 OD, that can quantitate colorimetrically stained gels.
80-6429-60 AD 107
5.3 Standardizing results
2-D electrophoresis is often used comparatively, and thus requires a reproducible method for determining relative
spot positions. Because precast Immobiline DryStrip gels are highly reproducible, the pI of a particular protein can
be estimated from its focusing position along a linear pH gradient Immobiline DryStrip gel. Detailed information on
Immobiline DryStrip pH gradients are found in the publication Immobiline DryStrip visualization of pH gradients (see
additional reading and reference material).
The second dimension can be calibrated using molecular weight marker proteins loaded to the side of the seconddimension gel. Often, there are abundant proteins in the sample for which the pI and molecular weight are known.
These proteins can serve as internal standards.
The pI of a protein is dependent on its chemical environment and can thus vary depending on the
experimental conditions used. The use of native pI markers is not recommended because they will run
differently in a native environment compared with a denaturing environment (e.g. urea).
5.4 Further analysis of protein spots
The procedure of picking and digesting spots can be performed manually or semi-automatically by manual transfer of
gels and microplates between the instruments as described below, or fully automatically in the integrated Ettan Spot
Handling Workstation.
Ettan Spot Handling Workstation comprises a stand-alone, controlled-atmosphere cabinet containing a spot picker/
spotter, digester, incubator, dryer, microplate hotel that also stores gel trays and MS targets, and robot for transferring
samples between the modules.
A computer with proprietary software controls the whole process. As an option, the processing in Ettan Spot
Handling Workstation can be integrated into Scierra™ Laboratory Workflow System (LWS), a communication platform
for the entire 2D-MS workflow. This software compiles and handles information, from receipt of samples, through
gel electrophoresis and processing in the workstation to information analysis and reporting. Communication and
information transfer from Ettan Spot Handling Workstation to Scierra LWS is completely automated.
5.4.1 Picking protein spots
Ettan Spot Picker is a robotic system that automatically picks selected protein spots from stained or destained gels
using a pick list created from the image analysis software, and transfers them into microplates.
DALT precast gels or lab-cast gels are stained with Coomassie, silver, or fluorescent dyes and two visible reference
markers are attached to each gel. The gels are scanned using ImageScanner or Typhoon and analyzed using
ImageMaster 2D Platinum or DeCyder 2-D Differential Analysis Software. The positions of selected protein spots
are exported as a pick list to Ettan Spot Picker. The gels are placed into the instrument under liquid and the camera
detects the reference markers. Control software converts spot pixel co-ordinates into picking co-ordinates, and the
Ettan Spot Picker selects and transfers gel plugs into 96-well microplates.
5.4.2 Digesting proteins and spotting onto MALDI-ToF MS slides
The gel plugs are digested in Ettan Digester, the supernatant peptides are mixed with matrix-assisted laser desorption
ionization time of flight mass spectrometry (MALDI-ToF MS) matrix material, and spotted onto MALDI-ToF MS slides
using Ettan Spotter.
5.4.3 MALDI-ToF mass spectrometry
Time-of-flight mass spectrometry is a technique for analyzing molecular weights based on the motion of ionized
samples in an electrical field. In Ettan MALDI-ToF Pro mass spectrometer, a matrix-bound sample is bombarded
with a pulsed laser beam to generate ions for subsequent detection. Ettan MALDI-ToF Pro provides fast and precise
identification of proteins in high-throughput peptide mass fingerprinting (PMF).
108 80-6429-60 AD
The novel quadratic field reflectron (Z2 reflectron) technology in Ettan MALDI-ToF Pro offers single run, post-source
decay (PSD) data acquisition in approximately 1 min. Automated database searching of PSD data allows rapid and
precise protein identification from single tryptic peptides.
In cases where PMF cannot provide unambiguous protein identification, reliable information can be obtained by
using the instrument in conjunction with CAF MALDI Sequencing Kit. Chemically assisted fragmentation (CAF) used
in conjunction with MALDI MS is a method for improving fragmentation of tryptic peptides by PSD. The technique
introduces a negative charge at the amino terminus of the peptide. Following fragmentation, only y ions (containing
C termini) are acquired in the spectra, while the neutralized b ions (containing N termini) are not observed. The spectra
containing y ions are easy to interpret and amino acid sequences can be deduced by calculating the mass differences
between the fragmented ions.
The software for automated PSD analysis has automated PSD data acquisition of selected peaks and automated PSD
spectrum processing and identification. This enables rapid and sensitive peptide sequencing and protein identification.
Chemically assisted fragmentation MALDI simplifies the amino acid sequencing of peptides and identification of
phosphorylation sites.
80-6429-60 AD 109
110 80-6429-60 AD
6. 2-D Fluorescence Difference Gel
Electrophoresis (2-D DIGE)
6.0 Overview
2-D Fluorescence Difference Gel Electrophoresis (2-D DIGE) is a method that labels protein samples prior to 2-D
electrophoresis, enabling accurate analysis of differences in protein abundance between samples (86). It is possible
to separate up to three different samples within the same 2-D gel (Fig 58). The technology is based on the specific
properties of spectrally resolvable dyes, CyDye DIGE Fluor dyes. Two sets of dyes are available—Cy™2, Cy3, and Cy5
minimal dyes, and Cy3 and Cy5 saturation dyes—that have been designed to be both mass- and charge-matched. As a
consequence, identical proteins labeled with each of the CyDye DIGE Fluor dyes will migrate to the same position on
a 2-D gel. This ability to separate more than one sample on a single gel permits the inclusion of up to two samples
and an internal standard (internal reference) in every gel. The internal standard is prepared by mixing together equal
amounts of each sample in the experiment and including this mixture on each gel.
CyDye DIGE Fluors are:
• Size- and charge-matched. The same labeled protein from different samples will migrate to the same position,
regardless of the dye used.
• pH insensitive. No change in signal over the wide pH range used during first-dimension separation (IEF) and
equivalent migration in SDS gels.
• Spectrally resolvable. The distinct signal from each fluor contributes to the accuracy.
• Highly sensitive and bright.
• Photostable. There is minimal loss of signal during labeling, separation, and scanning.
Fig 58. Multiplexing using the CyDye DIGE Fluor minimal dye option with Ettan DIGE system.
80-6429-60 AD 111
CyDye DIGE Fluors are available as minimal and saturation labeling dyes. The minimal dyes are intended for general 2-D
application use where sufficient amounts of sample are available. The saturation dyes, included in the scarce sample
and preparative gel labeling kits, are designed to be used for applications where only small amounts of sample are
available, for example in Laser Capture Microdissection.
Ettan DIGE system capitalizes on the ability to multiplex by combining CyDye DIGE Fluor dyes with DeCyder 2-D
Differential Analysis Software. DeCyder 2-D software has been designed specifically for Ettan DIGE applications. It
utilizes a proprietary co-detection algorithm that permits automatic detection, background subtraction, quantitation,
normalization, and inter-gel matching of the fluorescent images. The use of an internal standard gives an increased
confidence that the results reflect true biological effects and are not due to system variation.
The system comprises CyDye DIGE Fluor dyes for protein labeling; a choice of Ettan IPGphor 3 Isoelectric Focusing
System or Multiphor II Electrophoresis System for first-dimension separation; Ettan DALTsix, DALTtwelve, or SE 600
Ruby vertical electrophoresis systems for second-dimension separation; Typhoon Variable Mode Imager for advanced
imaging; and DeCyder 2-D Differential Analysis Software for quantitation and statistical analysis of protein differences
over a linear dynamic range of up to four orders of magnitude. Figure 59 summarizes the steps in the analysis.
1. Sample preparation
Proteins are extracted from cells or tissues of interest.
2. Sample labeling with CyDye DIGE fluors
Size and charge matched, spectrally resolvable
dyes enable simultaneous co-separation and
analysis of samples on a single gel–multiplexing.
4. 2-D electrophoresis
Up to three samples (one of which is
the internal pooled standard) can be simultaneously separated on a single
2-D gel, using IPGphor or Multiphor II
in the first dimension and Ettan DALT
in the second dimension.
5. Image acquisition
The gel is scanned using the highly sensitive
Typhoon FLA 9000, optimized for Ettan DIGE.
Perpendicular scanning assures quantitation
of protein expression levels and identification
of spots with high precision.
3. Addition of DTT and ampholytes
Following sample labeling, DTT and ampholytes are added to each sample.
6. Image analysis
DeCyder software automatically locates and analyzes
multiplexed samples in a gel within minutes. It allows
complex analysis of multiple gels to provide comparative
and accurate measurement of differential protein
expression.
Fig 59. Ettan DIGE system for protein analysis. Protein samples are extracted (1) and labeled with CyDye DIGE Fluor minimal dyes in the
absence of DTT and ampholytes (2). These are added following the labeling reaction (3). The fluors enable up to two samples plus an
internal standard to be resolved on the same 2-D gel (4). Gel images are obtained from the Typhoon Variable Mode Imager, which has
been optimized for use with CyDye DIGE Fluor minimal dyes (5). The images are automatically analyzed within DeCyder 2-D Differential
Analysis Software (6).
112 80-6429-60 AD
6.1 CyDye DIGE Fluor dyes
6.1.1 CyDye DIGE Fluor minimal dyes
CyDye DIGE Fluor minimal dyes consist of three bright, spectrally resolvable fluors (Cy2, Cy3, and Cy5) that are
matched for mass and charge. The fluors offer great sensitivity, detecting as little as 125 pg of transferrin and giving
a linear response to protein concentration of up to four orders of magnitude. In comparison, silver stain detects 1-60
ng of protein with less than a hundred-fold dynamic range (90, 91). Narrow excitation and emission bands mean that
the CyDye Fluor minimal dyes are spectrally distinct, which makes them ideal for multi-color detection (Fig 60). Most
importantly, the fluors are mass- and charge-matched so that the same protein labeled with any of the CyDye DIGE
Fluor minimal dyes will migrate to the same position within a 2-D gel (92–94). The novel properties of the CyDye DIGE
Fluor minimal dyes make them ideal for multiplexing different protein samples within the same 2-D gel. This permits
inclusion of an internal standard within each gel, which limits experimental variation and ensures accurate intra- and
inter-gel matching.
λex488nm λem520 BP40 Filters
λex532nm λem580 BP30 Filters
λex633nm λem670 BP30 Filters
Black curve, excitation; blue curve, emission for each of the dyes.
The numbers at the top of each curve indicate the maxima.
Cy2 excitation wavelength; Cy3 excitation wavelength;
Cy5 excitation (excitation wavelength = X nm ± 0.1 nm);
Cy2 emission filter; Cy3 emission filter; Cy5 emission filter.
Filters: the values for each of the excitation and emission filters
for each dye are chosen to minimize cross-talk, BP40: bandpass
of 40 = λem ± 20 nm; BP30: bandpass of 30 = λem ± 15 nm.
Fig 60. Excitation and emission spectra of CyDye DIGE Fluor minimal dyes Cy2, Cy3, and Cy5 showing the excitation and emission filters.
80-6429-60 AD 113
6.1.2 Minimal labeling of protein with CyDye DIGE Fluor minimal dyes
CyDye DIGE Fluor minimal dyes contain an N-hydroxysuccinimidyl ester reactive group. This enables labeling of lysine
residues within proteins, forming a covalent bond with the epsilon amino group of lysine residues to yield an amide
linkage (Fig 61). The recommended concentration of fluor present in a protein labeling reaction ensures that the fluor
is limiting. This leads to the labeling of approximately 1–2% of lysine residues. As a result, CyDye DIGE Fluor minimal
dyes will label only a small proportion of each protein in a sample, hence the expression “minimal labeling.”
The lysine amino acid in proteins carries an intrinsic positive charge which, when a CyDye DIGE Fluor minimal dye is
coupled to the lysine, replaces the lysine’s single positive charge with its own, ensuring that the pI of the protein does
not change.
When coupled to the protein, CyDye DIGE Fluor minimal dyes add approximately Mr 500 to the protein’s mass.
However, proteins should not be picked using the CyDye-labeled gel image as a positional reference due to slight
migration differences between the unlabeled and labeled proteins. These differences are due to the addition of a
single CyDye molecule to the labeled protein, which decreases the mobility of the protein with respect to unlabeled
protein. This effect is more marked for lower-molecular-weight proteins.
To ensure that the majority of unlabeled protein is picked, and therefore that sufficient protein is available for
identification by mass spectrometry, any gel for picking (usually a preparative gel) can be post-stained with a suitable
stain such as Deep Purple (see section 5.0 and appendix IV). The resulting gel is matched to the analytical set of DIGE
gels within the DeCyder 2-D Differential Analysis Software.
Fluor
linker
NHS ester
reactive group
Fluor
+ protein
@ pH 8.5
Lysine
O
N
H
NH
O
Fig 61. Schematic of the labeling reaction. CyDye DIGE Fluor containing NHS-ester active group covalently binds to lysine residue of
protein via an amide linkage.
Minimal labeling of proteins does not affect the mass spectrometry data used to identify proteins because only
1–2% of lysine residues are labeled, such that 98% of protein is unlabeled.
6.2 CyDye DIGE Fluor labeling kits with saturation
dyes for labeling scarce samples and preparative gels
Two labeling kits for scarce samples are available; one contains the Cy3 and Cy5 saturation dyes and the other
contains an additional vial of Cy3 dye to label a preparative gel. Each kit contains sufficient dye for at least 12
labeling reactions and allows labeling of as little as 5 µg of protein per labeling reaction, compared with 50 µg with
the minimal dyes. The additional vial of Cy3 dye contained in the CyDye DIGE Fluor Labeling Kit for Scarce Samples
and Preparative Gel Labeling allows for labeling up to 500 µg of protein. The saturation dyes Cy3 and Cy5 retain the
advantages described in section 6.1 for the minimal dyes.
The maleimide reactive group of the saturation dyes covalently bonds to the thiol group of cysteine residues of
proteins via a thioether linkage. To achieve maximal labeling of cysteine residues, the protocol uses a high fluor to
protein labeling ratio. This type of labeling method labels all available cysteines on each protein under the conditions
used, resulting in the majority of cysteine groups in a protein from a sample being labeled. For this reason, the method
has been called “saturation labeling.” See Figure 62 for an overview of the workflow and Figure 63 for a diagram of
the labeling process.
114 80-6429-60 AD
Fig 62. Outline of the Ettan DIGE system workflow for saturation labeling.
S
S
Protein
1. TCEP
37 °C, 1 h, dark
HS
HS
Dye
Protein
O
2. 37 °C, 30 min, dark
3. 2× sample buffer
O
N
H
N
O
O
O
N
H
O
Dy
e
Dye
S
S
N
O
N
H
Protein
O
N
O
Fig 63. Schematic of labeling reaction between CyDye DIGE Fluor saturation dye and the cysteine residues of a protein.
80-6429-60 AD 115
6.3 Ettan DIGE system workflow
The main steps in the Ettan DIGE system workflow are outlined in Figure 64.
There are several key differences between standard 2-D electrophoresis and Ettan DIGE system experiments. Failure
to incorporate these changes into an Ettan DIGE system experiment will impact upon data quality. See Table 41 for
these differences.
Experimental
design
Sample
preparation
1st and 2nd
dimension
separation
Determine
protein
concentration
Labeling
Post-stain gel
(e.g. Deep Purple
Total Protein Stain)
Sample evaluation/optimization
- Check lysate labeled with one
dye on 1-D gel
- Check three aliquots of the
internal standard, each labeled
with a different dye, on a 2-D gel
1st and 2nd
dimension
separation
Imaging
Preparative
gel workflow
DeCyder
analysis
Differential
expression
data
Analytical
gel workflow
Fig 64. Ettan DIGE system workflow.
116 80-6429-60 AD
Spot pick
list
Gel
processing
MALDI-MS
protein
identification
6.3.1 Experimental design for Ettan DIGE system applications
The differences in methodology between 2-D DIGE and traditional 2-D electrophoresis are outlined in Table 41.
Table 41. Differences in methodology between 2-D DIGE and traditional 2-D electrophoresis.
Step
2-D DIGE
Traditional 2-D electrophoresis
Sample preparation
Exclude carrier ampholyte and reductant until after labeling.
Carrier ampholyte and reductant included.
Concentrate protein to 1–10 mg/ml.
Concentration of sample is optional.
Labeling
Addition of CyDye required.
No CyDye required.
Labeling reaction terminated with addition of lysine.
No lysine addition required.
2× sample buffer (added to sample after labeling)
The sample buffer is made with 2× concentration of reductant
and carrier ampholyte.
(An equal volume of this buffer is
added to the labeled proteins.)
See the protocol in section 6.3.4.
Not required.
Electrophoresis
Use low-fluorescence glass plates.
Use standard glass plates.
Traditional 2-D electrophoresis suffers from two main types of variation:
System variation may arise due to differences in electrophoretic conditions between different gels, user-to-user
variation, or poor performance of the image analysis software. This variation can be controlled by the inclusion of an
internal standard within each gel and has also been minimized by development of the co-detection algorithm within
DeCyder 2-D Differential Analysis Software.
Inherent biological variations are differences that arise between different individuals, cell cultures, etc. These cannot
be removed from the analysis but can be accurately measured, and therefore differentiated from the system variation.
Inherent biological variation must be considered if genuine, induced biological changes (biologically significant
changes that arise as a consequence of the test conditions) are to be identified. It is strongly advised that biological
replicates (such as multiple cultures) be incorporated into the experimental design. The more biological replicates
included in the experiment, the more likely that inherent biological variation is taken into account, enabling a reliable
measure of significant induced biological change. Since the system variation with Ettan DIGE system is low due to the
internal standard and analysis method, biological variation will far exceed the system variation, and gel replicates are
therefore not necessary.
Experimental setup
To maximize the benefits of Ettan DIGE system, an internal standard should be incorporated within each gel. The ideal
internal standard comprises pooled aliquots from all the biological samples within the experiment. The internal standard
is labeled with one CyDye DIGE Fluor minimal dye (e.g. Cy2) and is run on every gel together with experimental
samples labeled with Cy3 or Cy5 CyDye DIGE Fluor minimal dyes (Table 42). This ensures that every spot on every gel is
represented within the common internal standard. Each protein spot in a sample can therefore be compared with its
representative within the internal standard to generate a ratio of relative expression (Fig 65).
Experimental design for using the two saturation dyes from the CyDye DIGE Fluor Labeling Kit for Scarce Samples and
Preparative Gel Labeling is simple: one dye is selected to label the internal standard (e.g. Cy3) and the other to label
the individual samples in the experiment.
80-6429-60 AD 117
Gel 1
Gel 2
Sample 2 – Cy5
Sample 4 – Cy5
Sample 1 – Cy3
Sample 3 – Cy3
Standard – Cy2
Standard – Cy2
Protein N (sample 1) : Protein N (standard gel 1)
Protein N (sample 2) : Protein N (standard gel 1)
Protein N (sample 3) : Protein N (standard gel 2)
Protein N (sample 4) : Protein N (standard gel 2)
Fig 65. Quantitation of protein abundance using co-detection algorithms. From each gel, three scan images are generated, Cy2 for the
internal standard, and Cy3 or Cy5 for test samples. The protein abundance of each spot in each sample is expressed as a normalized
ratio relative to spots from the in-gel internal standard.
The same internal standard is run on all gels within the experimental series. This creates an intrinsic link between
internal standard and samples in each gel, matching the internal standards between gels. Quantitative comparisons of
samples between gels are made based on the relative change of sample to its in-gel internal standard. This removes
inter-gel variation, a common problem associated with traditional 2-D, and separates gel-to-gel variation from
biological variation (Fig 66), enabling accurate, statistical quantitation of induced biological change between samples.
Ettan DIGE system is currently the only protein difference analysis technique to utilize this approach (95).
Gel 1
Gel 2
Sample 2 – Cy5
Sample 4 – Cy5
Sample 1 – Cy3
Sample 3 – Cy3
Standard – Cy2
Standard – Cy2
Matching and comparison of
samples using the relative
measure of sample to standard
Protein N in (sample 1) : (sample 2) : (sample 3) : (sample 4)
Fig 66. Matching and comparison of samples across gels. The internal standard sample, present on every gel, is used to aid matching
of spot patterns across all gels. The relative ratios of individual sample spots to their internal standards are used to accurately compare
protein abundance between samples on different gels.
The advantages of linking every sample in-gel to a common standard are:
• Accurate quantitation and accurate spot statistics between gels
• Increased confidence in matching between gels
• Flexibility of statistical analysis depending on the relationship between samples
• Separation of system variation from biological variation
118 80-6429-60 AD
Table 42 shows an example of a recommended experimental setup that was designed to derive statistical data on
differences between control and three treatment regimens A, B, and C. For the control and each treatment regimen,
four biological replicates were included. The internal standard (a pool of all samples: four control and 12 treated) was
labeled with CyDye DIGE Fluor Cy2 minimal dye and run on every gel.
Table 42. Setup for an Ettan DIGE system experiment.
Gel number
Cy2
Cy3
Cy5
1
Pooled standard
Control 1
Sample A3
2
Pooled standard
Sample A1
Sample B3
3
Pooled standard
Sample B1
Sample C3
4
Pooled standard
Sample C1
Control 3
5
Pooled standard
Control 2
Sample B4
6
Pooled standard
Sample A2
Sample C4
7
Pooled standard
Sample B2
Control 4
8
Pooled standard
Sample C2
Sample A4
Each gel contains CyDye DIGE Fluor Cy2 minimal dye-labeled standard, which is a pool of aliquots taken from each sample. Four biological
replicates (1–4) have been included for control and treated (A, B, or C) samples. The samples have been arranged between gels to
ensure an even distribution between those labeled with CyDye DIGE Fluor Cy3 minimal dye and those labeled with CyDye DIGE Fluor
Cy5 minimal dye. This setup avoids repeatedly linking the same two treatment types on multiple gels. For further information relating to
experimental design, please refer to the Ettan DIGE user manual.
6.3.2 Sample preparation for Ettan DIGE system applications
CyDye DIGE Fluor minimal dyes, used for protein labeling in Ettan DIGE system applications, form a peptide linkage
between the fluor and lysine residues within the protein. Components such as primary amines (e.g. ampholytes) will
compete with the proteins for fluor binding. Thiols (e.g. DTT) also cause a reduction in labeling efficiency. The result
will be fewer fluor-labeled proteins, which may affect the data after scanning and spot detection. To achieve optimal
labeling, such components should be omitted from both the lysis and sample buffers and are only added to the
sample after labeling.
The labeling reaction with CyDye DIGE Fluor minimal dyes is most efficient at pH 8.5. Below pH 8.0, reactivity of the
label is reduced; above pH 9.0, increased non-specific binding to thiol groups is promoted and the NHS ester may
become inactivated due to hydrolysis. Lysis and sample solutions should be buffered using NaOH to pH 8.5.
CyDye DIGE Fluor saturation dyes, included in the labeling kits for scarce samples and preparative gels, covalently
bind to the thiol group of cysteine residues via a thioether linkage. The labeling reaction with CyDye DIGE Fluor
saturation dyes is most efficient at pH 8.0.
For further information on compatible reagents for labeling, please refer to appendix E.2 of the Ettan DIGE user manual.
Protocol for preparing protein from cell cultures and then labeling with
CyDye DIGE Fluor minimal dyes
A. Washing cells
If using a cell culture, wash cells to remove any growth media or reagents that might affect the CyDye DIGE Fluor
minimal dye labeling process. Check that the cell wash buffer does not contain any primary amines or thiols that may
interfere with the downstream labeling process.
DIGE cell washing buffer contains 10 mM Tris pH 8.0, 5 mM magnesium acetate.
DIGE cell lysis buffer contains 30 mM Tris, 2 M thiourea, 7 M urea, 4% CHAPS (w/v) at pH 8.5.
B. Lysing cells in lysis buffer
1. Resuspend the washed cell pellet in 1 ml of DIGE cell lysis buffer at pH 8.5 and leave on ice for 10 min
(approximately 4 x 1010 E. coli cells will yield 5–10 mg protein).
2. Lyse cells (see section 1.1) on ice until solution becomes less cloudy.
3. Centrifuge to pellet cell debris.
80-6429-60 AD 119
4. Transfer supernatant into a tube and if necessary adjust to pH 8.5.
5. Determine protein concentration. For best results the sample concentration should be 1–10 mg/ml.
For further information relating to sample preparation, please refer to the Ettan DIGE user manual.
If working with a sample in an unknown buffer, the sample should be precipitated and resuspended in an Ettan DIGE
system-compatible buffer. 2-D Clean-Up Kit can be used for this purpose (see section 1.4.1).
6.3.3 Sample labeling with minimal dyes for Ettan DIGE system applications
With CyDye DIGE Fluor minimal dyes, it is important that primary amines (e.g. ampholytes) and thiols (e.g. DTT) are
excluded from the sample until after labeling with the dyes has been completed.
For best results the sample concentration should be 1–10 mg/ml (5 mg/ml is optimal). For efficient labeling, the pH of
the labeling reaction should be between 8.0 and 9.0 (pH 8.5 is optimal).
A. Preparation of CyDye DIGE Fluor minimal dyes for protein labeling
The dimethylformamide (DMF) used to reconstitute the fluors should be high-quality anhydrous
(< 0.005% H2O, > 99.8% pure). It must not become contaminated with water, which will start to degrade the
DMF to amine compounds. The DMF stock solution should be replaced at least every 3 months.
Use of molecular sieves will help keep DMF in an anhydrous condition.
Condensation should be prevented from forming within the fluor vials. Once removed from the freezer, the fluor tubes
should be left for 5 min to equilibrate to room temperature prior to opening.
Although CyDye DIGE Fluor minimal dyes and labeled proteins are very photostable, the fluors and labeled proteins
should be kept covered or in the dark.
1. Reconstitute CyDye DIGE Fluor minimal dyes
Once the fluors have equilibrated to room temperature, dispense the specified volume of DMF into the fluor vial to
achieve a concentration of 1 nmol/µl (see specification sheet supplied with the fluor), e.g. add 25 µl DMF to 25 nmol of
fluor. Mix vigorously and centrifuge to collect fluor at the bottom of the vial. The concentrated stock solution is stable
for two months at -20 °C or until the expiry date if sooner.
2. Dilute CyDye DIGE Fluor minimal dyes to a working stock concentration
Dilute the concentrated stock solutions to a working fluor concentration of 400 pmol/µl using DMF, e.g. add 2 µl of
concentrated stock fluor to 3 µl of DMF.
The working fluor solution is stable for one week at -20 °C or until the expiry date if sooner.
B. Labeling protein sample with CyDye DIGE Fluor minimal dyes.
A pooled internal standard should be created from all of the samples. This will need to be sufficient for inclusion on
every gel.
It is recommended that the ratio of protein to fluor is maintained at 50 µg protein to 400 pmol fluor. However, it may
be necessary to determine the optimum ratio for individual samples.
1. Label proteins
Add 1 µl of working fluor solution (400 pmol/µl) to a volume of sample containing 50 µg of protein. Mix thoroughly by
vortexing. Centrifuge to collect labeling mixture at the bottom of the tube. Incubate on ice for 30 min in the dark.
2. Quench the labeling reaction
Add 1 µl of 10 mM lysine to stop the labeling reaction. Mix well and leave on ice for 10 min in the dark.
3. Store sample
The labeled sample can either be processed immediately or stored for up to three months at -70 °C in the dark.
To confirm efficient labeling, any new protein samples should be labeled with CyDye DIGE Fluor Cy5 minimal dye
and run on a 1-D SDS-PAGE gel to compare the efficiency of labeling against a control lysate already known to label
successfully. A lysate of known concentration in an Ettan DIGE system-compatible lysis buffer would be a suitable
alternative control.
120 80-6429-60 AD
6.3.4 Two-dimensional separation of protein samples
Separation of labeled proteins is carried out using traditional 2-D polyacrylamide gel electrophoresis (see chapters 2–4).
Protocol
A. Combining protein samples for multiplexing
Protein samples labeled with different CyDye DIGE Fluor minimal dyes are combined according to the experimental
design (see section 6.3). For best results, one or two labeled protein samples (usually Cy3 or Cy5) are combined with a
labeled internal standard (usually Cy2), which is a pool of aliquots of all biological samples in the experiment.
B. Diluting labeled protein sample in sample buffer
The sample mixture is diluted further in sample buffer prior to separating the individual proteins on a 2-D gel.
Ettan DIGE system-compatible 2× sample buffer contains 7 M urea, 2 M thiourea, 2% CHAPS (w/v), 2% IPG buffer or
Pharmalyte (v/v) for IEF, 2% DTT (w/v).
Add 1 volume of 2× sample buffer to sample. Mix and leave on ice for at least 10 min.
C. Rehydrating Immobiline DryStrip gel
Refer to sections 2.4–2.7 for a discussion of rehydration and sample application methods.
Ettan DIGE system-compatible rehydration buffer contains 7 M urea, 2 M thiourea, 4% CHAPS (w/v), 1% IPG buffer or
Pharmalyte (v/v) for IEF, 2% (w/v) DTT.
D. Separating proteins in the first dimension
Ettan IPGphor 3 Isoelectric Focusing System and Multiphor II Electrophoresis System are both suitable for Ettan DIGE
system applications. Detailed instructions for use of the systems are given in chapters 2 and 4, respectively. Ettan
DIGE applications are described in detail in the Ettan DIGE user manual.
E. Separating proteins in the second dimension
Low-fluorescence glass plates must be used for gels used in Ettan DIGE system. Standard glass or plastic-backed
plates can result in the generation of a high background signal.
DIGE Gel is a 12.5% precast, low-fluorescent polyacrylamide gel cast in a low-fluorescent glass cassette specially
developed for 2-D DIGE analysis. DIGE Gel should be used with the DIGE Buffer Kit, which consists of concentrated
running buffers and Sealing Solution for attaching Immobiline DryStrip Gels (IPG strips) to the top of the
polyacrylamide gel. The capacity of the buffer system is similar to the commonly used Laemmli (Tris-Glycine) buffer
system, and the separation performance of DIGE Gel is comparable to 12.5% Laemmli gels.
DALT gels are large enough to accommodate the longest Immobiline DryStrip gels (24 cm) and can be run in batches of
up to 12 gels at a time. DALT gels can also be poured using the DALT gel caster. DALT gels can also be run using the
DIGE buffer kit. Detailed protocols for gel casting can be found in the Ettan DIGE user manual, Ettan DALTtwelve user
manual, and Ettan DALTsix user manual.
The procedures for equilibrating strips, positioning them, and electrophoresis are identical to those for standard 2-D
analysis. Refer to sections 3.1 and 3.3 for details.
If the gels are to be scanned immediately, store the gels in SDS electrophoresis running buffer at room
temperature in a light-tight container. Scan the gels as soon as possible as the protein spots in the gel will
diffuse with time. If the gels cannot be scanned on the day of running, they should be stored in the dark
at +4 °C and kept moist. Remember to let the gels warm to room temperature before scanning because
temperature affects the fluorescent signal.
Do not fix the gels prior to scanning as this will affect quantitation of the labeled protein spots.
80-6429-60 AD 121
6.3.5 Summary of key differences between minimal labeling
and saturation labeling
Table 43 lists the key differences between minimal labeling and saturation labeling.
Table 43. Comparison of minimal labeling and saturation labeling experiments.
Saturation labeling
Minimal labeling
Sample preparation
Cell lysis buffer is at pH 8.0.
Cell lysis buffer is at pH 8.5.
Dyes
Maleimide dyes.
NHS ester dyes.
Label cysteine residues.
Label lysine residues.
Two dyes available.
Three dyes available.
CyDye DIGE Fluor saturation dyes are Once reconstituted, the concentrated stock
reconstituted at 2 mM (analytical gels) (1 mM) of CyDye DIGE Fluor minimal dyes is
or 20 mM (preparative gels).
stable for up to 2 months at -15 °C to -30 °C.
Once reconstituted, the dyes are stable The working concentration of the dyes is
for up to 2 months at -15 °C to -30 °C.
0.4 mM and is stable for 1 week.
Once reconstituted, dyes do not need
to be diluted further. Reducing step
Proteins must be reduced using TCEP prior to labeling.
No reduction step required.
Protein labeling
Labeling reaction performed at 37 °C.
Labeling reaction quenched using 2x sample buffer.
Labeling is optimized by titrating TCEP and dye (Cy3 and Cy5) then analyzing on a 1-D gel.
Labeled proteins are stable for 1 month at -70 °C.
Labeling reaction performed at 4 °C.
Labeling reaction quenched with
10 mM lysine.
Labeling is optimized by comparing labeled
samples on a 1-D gel.
Labeled proteins have stability equivalent
to unlabeled protein at -70 °C.
Protein separation and analysis
No iodoacetamide equilibration step prior to 2-D electrophoresis.
A Cy3 labeled sample is used to prepare a preparative gel for spot picking. No staining is required.
Iodoacetamide equilibration step required.
An unlabeled sample is used to prepare a
preparative gel for spot picking.
The gel must be stained using a fluorescent
post-stain to allow matching to analytical
gels for picking.
6.3.6 Imaging
Typhoon is a highly sensitive variable-mode imager that has been adapted to meet the specific needs of 2-D DIGE.
Typhoon Variable Mode Imager optimally detects Cy2, Cy3, and Cy5 signals with exceptional signal to noise ratio
due to consistent point-light illumination that eliminates the need for image stitching, confocal optics that exclude
artifacts, and narrow band-pass filters that maximize signal to noise ratio (Fig 60).
A linear protein concentration response over five orders of magnitude is possible with Ettan DIGE system (96)
compared with silver, which has a dynamic range of less than two orders of magnitude (97). In addition, silver-stained
proteins saturate more readily, which produces data that cannot be accurately quantitated. The wide dynamic range
provided by CyDye 2-D DIGE Fluor minimal dyes, in combination with the Typhoon Variable Mode Imager, enables
production of data that is quantitative and reproducible, and that offers a sensitivity down to
125 pg protein, compared with approximately 5 ng for silver staining (98).
Specific gel-alignment guides enable the correct positioning of both DALT and SE 600 Ruby gels on the scanner to
simplify gel handling and to reduce hands-on time. Two large-format DALT gels or four SE 600 Ruby gels can be
imaged simultaneously, and the file outputs are separated automatically in a format that is compatible with DeCyder
2-D Differential Analysis Software. The gels can be easily scanned between low-fluorescence glass plates, which
prevents drying and shrinkage, and also allows for further running and scanning.
For additional information relating to the use of the Typhoon Variable Mode Imager for the Ettan DIGE system, refer to
the Ettan DIGE user manual.
122 80-6429-60 AD
6.3.7 Image analysis with DeCyder 2-D Differential Analysis Software
DeCyder 2-D Differential Analysis Software, developed to exploit the advantages of CyDye DIGE Fluor dyes, consists of a
fully automated image analysis software suite that enables the detection, quantitation, matching, and analysis
of gels used with Ettan DIGE system.
The co-detection algorithm in DeCyder 2-D software co-detects overlaid image pairs and produces identical spot
boundaries for each pair. This enables direct spot volume ratio measurements and therefore produces an accurate
comparison of every protein with its representative in-gel internal standard. The software automatically performs
detection, background subtraction, quantitation, and normalization, which takes into account any differences in the
dyes, i.e. molar extinction co-efficients, quantum yields, etc.
These steps can be broken into the following processes, which form part of the built-in functionality of
DeCyder 2-D software, and are performed automatically with minimum user intervention:
• Spot detection
• Background subtraction
• In-gel normalization
• Gel artifact removal
• Gel-to-gel matching
• Statistical analysis
DeCyder 2-D software utilizes the inclusion of an internal standard within each gel by performing gel-to-gel
matching on the standard samples only. The presence of the same standard sample on every gel enables accurate
normalization of the individual samples, decreasing gel-to-gel and software analysis variation. Differences in
expression of less than 10%, with over 95% confidence, can be achieved within minutes. In conjunction with CyDye
DIGE Fluor dyes, DeCyder 2-D software allows gel analysis using different experimental designs with various degrees of
complexity. A simple control-treated experiment, through to a multi-factorial experiment addressing factors such as
dose and time, can be performed in a single analysis.
The DeCyder 2-D software suite consists of several modules:
• Batch Processor
For automated detection, quantitation matching, and comparison of multiple gels used with Ettan DIGE system.
• Differential in-gel analysis (DIA)
For co-detection, background subtraction, normalization, and quantitation of spots in an image pair.
• Biological variation analysis (BVA)
For matching multiple gels for comparison and statistical analysis of protein-abundance changes.
• XML Toolbox
For exporting spot data from DIA or BVA modules for further analysis.
For further information relating to DeCyder 2-D Differential Analysis Software, please refer to the Ettan DIGE
user manual.
• Extended Data Analysis (EDA)
Multivariate analysis of data from several BVA workspaces.
6.3.8 Further analysis of protein spots
Ettan DIGE system is fully compatible with mass spectrometry analysis and has been fully integrated into the Ettan
proteomics platform. DeCyder 2-D software will generate a pick-list of spots of interest that can be exported directly into
Ettan Spot Picker or Ettan Spot Handling Workstation.
Protein spots are automatically picked from the glass-backed gel. For backing of gels to glass, see appendix V.
Although spots can be picked directly from post-stained analytical gels, where possible, preparative-scale gels
provide more material for analysis by mass spectrometry. A preparative gel, post-stained, can be matched to previous
analytical gels by DeCyder 2-D Differential Analysis Software.
See also section 5.4.
80-6429-60 AD 123
6.4 Troubleshooting 2-D DIGE
For troubleshooting 2-D DIGE results, please refer to the Ettan DIGE user manual.
124 80-6429-60 AD
7. Troubleshooting
Table 44 lists problems that may be encountered in 2-D electrophoresis results, describes the possible causes,
and suggests ways to prevent problems in future experiments.
For troubleshooting problems encountered during the various steps of the 2-D process, refer to the following:
• Table 20, page 70. Troubleshooting first-dimension IEF: Ettan IPGphor 3 Isoelectric Focusing System.
• Table 21, page 71. Troubleshooting first-dimension IEF: Employing the Manifold.
• Table 34, page 90. Troubleshooting vertical second-dimension SDS-PAGE.
• Table 36, page 95. Troubleshooting Immobiline DryStrip gel rehydration in Reswelling Tray.
• Table 38, page 103. Troubleshooting first-dimension IEF: Multiphor II Electrophoresis System and
Immobiline DryStrip Kit.
• Table 40, page 107. Troubleshooting second-dimension SDS: Multiphor II Electrophoresis System.
For troubleshooting 2-D DIGE results, please refer to the Ettan DIGE user manual.
Table 44. Troubleshooting 2-D results.
Symptom
Possible cause
Remedy
No distinct spots are visible
Sample is insufficient.
Insufficient sample entered the Immobiline DryStrip gel
due to poor sample solubilization.
Increase the amount of sample applied.
Increase the concentration of the solubilizing
components in the sample solution
(see section 1.6).
Sample contains impurities
that prevent focusing.
Increase the focusing time or modify the
sample preparation method (see chapter 1).
The pH gradient is
incorrectly oriented.
The “+” end of the Immobiline DryStrip is the
acidic end and should point toward the
anode (+).
(Flatbed gel format) Immobiline DryStrip gel is
placed wrong side down on second-dimension gel.
Ensure that the Immobiline DryStrip gel
is placed gel-side down (plastic backing
upward) on the SDS second-dimension gel.
Detection method was not
sensitive enough.
Use another detection method (e.g. silver staining instead of Coomassie blue staining).
Failure of detection reagents. Check expiry dates on staining solutions. Prepare fresh staining solutions.
Individual proteins appear as multiple Protein carbamylation.
spots or are missing, unclear, or in the wrong position
Do not heat any solutions containing urea
above 30 ºC, as cyanate, a urea degradation
product, will carbamylate proteins, changing
their pI.
Protein oxidation.
Use DeStreak Rehydration Solution.
During equilibration, add DTT in first step to
reduce the disulfide. Add iodoacetamide in
the second step to alkylate the thiol groups
to prevent proteins from reoxidizing.
continues on following page
80-6429-60 AD 125
Table 44. Troubleshooting 2-D results (continued).
Symptom
Possible cause
Remedy
Distortion of 2-D pattern
(Vertical gel format) The top
surface of the second-
dimension gel is not flat.
Immediately after pouring the gel, overlay
the surface with water-saturated 1-butanol.
(Vertical gel format) Uneven
polymerization of gel due to
incomplete polymerization,
too rapid polymerization,
or leakage during gel casting.
Polymerization can be accelerated by
increasing by 50% the amount of ammonium
persulfate and TEMED used. Polymerization
can be slowed by decreasing by 33% the
amount of ammonium persulfate and
TEMED used.
Ensure that there is no leakage during gel
casting.
Sample applied at too Horizontal streaking or incompletely focused spots (anodic sample application, acidic pH.
in which the problem is visible at the anodic end of the IPG strip)
Increase the concentration of IPG buffer
in sample and Immobiline DryStrip.
Add slightly more alkaline IPG buffer to
the sample.
Apply the sample at the cathode.
Note: Repeated precipitation resolubilization
cycles produce or increase horizontal
streaking.
See section 1.6 for general guidelines for
sample solubilization.
Horizontal streaking or incompletely Sample is poorly soluble in focused spots (rehydration loading)
rehydration solution.
Increase the concentration of the solubilizing
components in the rehydration solution
(see section 2.6).
Increase concentration of IPG Buffer.
Underfocusing. Focusing Prolong focusing time.
time was not long enough to
achieve steady-state focusing.
Horizontal streaking or incompletely focused Interfering substances.
Non-protein impurities in the spots (all sample application methods)
sample can interfere with IEF, causing horizontal streaking.
continues on following page
126 80-6429-60 AD
Degas the gel solution.
Modify sample preparation to limit these
contaminants (see section 1.4).
Use 2-D Clean-Up Kit (section 1.4.1).
The effect of ionic impurities can be reduced
by modifying the IEF protocol. Limit the
voltage to 100-150 V for 2 h, then resume a
normal voltage step program. This allows the
ions in the sample to move to the ends of the
Immobiline DryStrip gel.
Table 44. Troubleshooting 2-D results (continued).
Symptom
Possible cause
Remedy
Ionic detergent in sample.
If the ionic detergent SDS is used in sample
preparation, the final concentration must not
exceed 0.25% after dilution into the rehydration
solution. Additionally, the concentration of the
nonionic detergent present must be at least
eight times higher than the concentration of
any ionic detergent to ensure complete
removal of SDS from the protein.
Horizontal stripes across gel
Prepare fresh agarose overlay and
equilibration solution
Impurities in agarose overlay
or equilibration solution.
Prominent vertical streak at the point (Flatbed gel format) Sample
aggregation or precipitation.
of sample application (when loading Immobiline DryStrip gels and
sample cups)
Program a low initial voltage and increase
voltage gradually.
Vertical gap in 2-D pattern
Modify sample preparation. (See section 1.4).
Impurities in sample.
Dilute the sample and apply as a larger
volume.
Impurities in rehydration
solution components.
Deionize urea solutions.
Bubble between Immobiline
DryStrip gel and top surface
of second-dimension gel.
Ensure that no bubbles are trapped between
the Immobiline DryStrip gel and the top
surface of the second-dimension gel.
(Flatbed gel format) Urea
crystals on the surface of the
Immobiline DryStrip gel.
Allow residual equilibration solution to drain
from the Immobiline DryStrip gel before placing
the strip on the second-dimension gel.
(Flatbed gel format) Bubbles
under the Immobiline DryStrip gel.
Use only high-quality reagents.
Ensure that the Immobiline DryStrip gel is
placed firmly on the gel with no air bubbles
trapped underneath. Stroke the plastic
backing of the Immobiline DryStrip gel
gently with a pair of forceps to remove
trapped bubbles.
continues on following page
80-6429-60 AD 127
Table 44. Troubleshooting 2-D results (continued).
Symptom
Possible cause
Poor representation of higher-
Proteolysis of sample.
molecular-weight proteins
Remedy
Prepare sample in a manner that limits
proteolysis and/or use protease inhibitors
(see section 1.2).
Insufficient equilibration.
Prolong equilibration time.
Poor transfer of protein from Immobiline DryStrip gel to second-dimension gel.
Employ a low current sample entry phase
in the second-dimension electrophoresis run.
Poor entry of sample protein
during rehydration.
Use recommended volume of rehydration
solution (Table 18).
Point streaking
(Silver staining) Dirty plates
used to cast gel or particulate
material on the surface of the gel. DTT and other thiol-
reducing agents exacerbate
this effect.
Properly wash glass plates. Scavenge any
excess or residual thiol-reducing agent with
iodoacetamide before loading the Immobiline
DryStrip gel onto the second-dimension gel.
Background smear toward bottom of gel
(Silver or Coomassie blue staining) Staining of carrier ampholytes.
Use IPG Buffer as carrier ampholyte mixture.
Reduce concentration if necessary.
Prolong fixing time.
Background smear toward top of gel
(Silver staining) Nucleic acids
in sample.
Add DNase and RNase to hydrolyze
nucleic acids.
Note: The proteins DNase and RNase may appear on the 2-D map.
High background in top region of gel
Make fresh SDS electrophoresis buffer.
128 80-6429-60 AD
Protein contaminant in SDS
electrophoresis buffer or
dirty electrophoresis unit.
Clean electrophoresis unit.
Appendix I
Solutions
Some of the chemicals used in the procedures—acrylamide, N,N’-methylenebisacrylamide, ammonium
persulfate, TEMED, DTT, iodoacetamide, and DeStreak Reagent—are very hazardous. Acrylamide monomer,
for example, is a neurotoxin and suspected carcinogen. Read the manufacturer’s safety data sheet (MSDS)
detailing the properties and precautions for all chemicals in your laboratory. These safety data sheets should be
reviewed prior to starting the procedures described in this handbook. General handling procedures for hazardous chemicals include using double latex gloves for all protocols. Hazardous materials should be weighed in
a fume hood while wearing a disposable dust mask. Follow all local rules and regulations for handling and
disposal of materials.
A. Sample preparation solution (with urea) for 2-D electrophoresis
[8 M urea, 4% CHAPS, 2% Pharmalyte or IPG buffer (carrier ampholytes), 40 mM DTT, 25 ml]
Final concentration
Amount
Urea (FW 60.06)
8 M*
12 g
CHAPS†
4% (w/v)
1.0 g
Pharmalyte‡ or IPG Buffer§
2% (v/v)
500 µl
DTT (FW 154.2)
40 mM
154 mg
Double-distilled water
—
to 25 ml (16 ml required)
* If necessary, the concentration of urea can be increased to 9 or 9.8 M.
†
Other neutral or zwitterionic detergents may be used at concentrations up to 2% (w/v). Examples include Triton X-100, NP-40, octyl
glucoside, and the alkylamidosulfobetaine detergents ASB-14 and ASB-16 (Calbiochem).
‡
Carrier ampholytes (Pharmalyte or IPG buffer) and DTT should be excluded from the sample extraction solution if the samples are to
be labeled using 2-D DIGE. See Ettan DIGE User Manual for details.
§
Use IPG Buffer in the pH range corresponding to the pH range of the IEF separation to be performed, or Pharmalyte in a pH range
approximating the pH range of the IEF separation to be performed.
Store in 2.5-ml aliquots at -20 °C.
Note: Protease inhibitors may be added if necessary.
B. Sample preparation solution (with urea and thiourea)
for 2-D electrophoresis
[7 M urea, 2 M thiourea, 4% CHAPS, 2% Pharmalyte or IPG Buffer (carrier ampholytes), 40 mM DTT, 25 ml]
Final concentration
Amount
Urea (FW 60.06)
7 M
10.5 g
Thiourea (FW 76.12)
2 M
3.8 g
1.0 g
CHAPS*
4% (w/v)
Pharmalyte† or IPG Buffer‡
2% (v/v)
500 µl
DTT (FW 154.2)
40 mM
154 mg
Double-distilled water
—
to 25 ml (13.5 ml required)
* Other neutral or zwitterionic detergents may be used at concentrations up to 2% (w/v). Examples include Triton X-100, NP-40, octyl
glucoside, and the alkylamidosulfobetaine detergents ASB-14 and ASB-16 (Calbiochem).
†
Carrier ampholytes (Pharmalyte or IPG buffer) should be excluded from the sample extraction solution if the samples are to be
labeled using 2-D DIGE.
‡
Use IPG Buffer in the pH range corresponding to the pH range of the IEF separation to be performed, or Pharmalyte in a pH range
approximating the pH range of the IEF separation to be performed.
Store in 2.5-ml aliquots at -20 °C.
80-6429-60 AD 129
C. Urea rehydration stock solution
(8 M urea, 2% CHAPS, 0.5/2% Pharmalyte or IPG Buffer, 0.002% bromophenol blue, 25 ml)*
Final concentration
Urea (FW 60.06)
8 M†
Amount
12 g
CHAPS‡
2% (w/v)
0.5 g
Pharmalyte or IPG Buffer§
(same range as the IPG strip)
0.5% (v/v) or 2% (v/v)¶
125 µl or 500 µl
1% Bromophenol blue stock solution
0.002%
50 µl
Double-distilled water
—
to 25 ml (16 ml required)
* DTT is added just prior to use: 7 mg DTT per 2.5-ml aliquot of rehydration stock solution. For rehydration loading, sample is also
added to the aliquot of rehydration solution just prior to use.
†
If necessary, the concentration of urea can be increased to 9 M or 9.8 M.
‡
Other neutral or zwitterionic detergents may be used at concentrations up to 2% (w/v). Examples include Triton X-100, NP-40, octyl
glucoside, and the alkylamidosulfobetaine detergents ASB-14 and ASB-16 (Calbiochem).
§
As an alternative to IPG Buffer, use Pharmalyte 3–10 for Immobiline DryStrip 3–10 or 3–10 NL, Pharmalyte 5–8 for Immobiline
DryStrip 4–7.
¶
A Pharmalyte/IPG Buffer concentration of 0.5% (125 µl) is recommended with Ettan IPGphor 3 Isoelectric Focusing System and an
IPG Buffer/Pharmalyte concentration of 2% (500 µl) is recommended with the Multiphor II and Immobiline DryStrip Kit system.
Store in 2.5-ml aliquots at -20 °C.
D. Thiourea rehydration stock solution
(7 M urea, 2 M thiourea, 2% CHAPS, 0.5/2% Pharmalyte or IPG Buffer, 0.002% bromophenol blue, 25 ml)
Final concentration
Amount
Urea (FW 60.06)
7 M
10.5 g
Thiourea (FW 76.12)
2 M
3.8 g
CHAPS†
2% (w/v)
0.5 g
Pharmalyte or IPG Buffer
0.5% (v/v) or 2% (v/v)‡
125 µl or 500 µl
1% Bromophenol blue stock solution
0.002%
50 µl
Double-distilled water
—
to 25 ml (13.5 ml required)
* DTT is added just prior to use: Add 7 mg DTT per 2.5-ml aliquot of rehydration stock solution.
†
Other neutral or zwitterionic detergents may be used at concentrations up to 2% (w/v). Examples include Triton X-100, NP-40, octyl
glucoside, and the alkylamidosulfobetaine detergents ASB-14 and ASB-16 (Calbiochem).
‡
A Pharmalyte/IPG Buffer concentration of 0.5% (125 µl) is recommended with Ettan IPGphor 3 Isoelectric Focusing System and an
IPG Buffer/Pharmalyte concentration of 2% (500 µl) is recommended with the Multiphor II and Immobiline DryStrip Kit system.
Store in 2.5-ml aliquots at -20 °C.
E. SDS equilibration buffer solution
(6 M urea, 75 mM Tris-HCl pH 8.8, 29.3% glycerol, 2% SDS, 0.002% bromophenol blue, 200 ml)*
Final concentration
Amount
Urea (FW 60.06)
6 M
72.1 g
Tris-HCl, pH 8.8 (see solution H)
75 mM
10.0 ml
Glycerol (87% w/w)
29.3% (v/v)
69 ml (84.2 g)
SDS (FW 288.38)
2% (w/v)
4.0 g
1% Bromophenol blue stock solution
0.002% (w/v)
400 µl
Double-distilled water
—
to 200 ml
* This is a stock solution. Just prior to use, add DTT or iodoacetamide (for first or second equilibration, respectively) as described in the
protocol in section 3.1.2.
Store in 20- or 50-ml aliquots at -20 °C.
130 80-6429-60 AD
F. 10× Laemmli SDS electrophoresis buffer
(250 mM Tris base, 1.92 M glycine, 1% SDS, 10 l)*
Final concentration
Amount
Tris base (FW 121.1)
250 mM
303 g
Glycine (FW 75.07)
1.92 M
1441 g
SDS (FW 288.38)
1% (w/v)
100 g
Double-distilled water
—
to 10 l
* The pH of this solution should not be adjusted.
Store at room temperature.
See also Recipe M for 1× Laemmli SDS electrophoresis buffer.
G. 30% T, 2.6% C monomer stock solution
(30% acrylamide, 0.8% N,N’-methylenebisacrylamide, 1 l)
Final concentration
Amount
Acrylamide (FW 71.08)
30%
300 g
N,N’-methylenebisacrylamide (FW 154.17)
0.8%
8g
Double-distilled water
—
to 1 l
Filter solution through a 0.45-µm filter. Store at 4 °C in the dark.
H. 4× resolving gel buffer solution
(1.5 M Tris base, pH 8.8, 1 l)
Final concentration
Amount
Tris base (FW 121.1)
1.5 M
181.7 g
750 ml
Double-distilled water
—
HClaq —
adjust to pH 8.8
Double-distilled water
—
to 1 l
Filter solution through a 0.45-µm filter. Store at 4 °C.
I. Bromophenol blue stock solution
Final concentration
Amount
Bromophenol blue
1%
100 mg
Tris-base
50 mM
60 mg
Double-distilled water
—
to 10 ml
J. 10% SDS solution
(10% SDS, 50 ml)
Final concentration
Amount
SDS (FW 288.38)
10% (w/v)
5.0 g
Double-distilled water
—
to 50 ml
Filter solution through a 0.45-µm filter. Store at room temperature.
80-6429-60 AD 131
K. 10% ammonium persulfate solution
(10% ammonium persulfate, 10 ml and 1 ml)
Final concentration
Amount for 10 ml
Amount for 1 ml
Ammonium persulfate (FW 228.20)
10% (w/v)
1.0 g
0.1 g
Double-distilled water
—
to 10 ml
to 1 ml
Fresh ammonium persulfate “crackles” when water is added. If it does not, replace it with fresh stock. Prepare just
prior to use.
L. Gel storage solution
(375 mM Tris-HCl, 0.1% SDS, 1 l)
Final concentration
Amount
4× Resolving gel buffer (see solution H above)
1×
250 ml
10% SDS (see solution J above)
0.1%
10 ml
Double-distilled water
—
to 1 l
Store at 4 °C.
M. 1× Laemmli SDS electrophoresis buffer
(25 mM Tris base, 192 mM glycine, 0.1% SDS, 10 l)*
Final concentration
Amount
Tris base (FW 121.1)
25 mM
30.3 g
Glycine (FW 75.07)
192 mM
144.0 g
SDS (FW 288.38)
0.1% (w/v)
10.0 g
Double-distilled water
—
to 10 l
* The pH of this solution should not be adjusted.
This solution can be prepared by diluting one volume of 10× Laemmli SDS buffer (solution F) with nine volumes of
double-distilled water.
Store at room temperature.
N. Agarose sealing solution
(25 mM Tris base, 192 mM glycine, 0.1% SDS, 0.5% agarose, 0.002% bromophenol blue, 100 ml)
Final concentration
Amount
Laemmli SDS electrophoresis buffer
(see solution M)
100 ml
Agarose (NA or M)
0.5%
0.5 g
1% Bromophenol blue stock solution
0.002% (w/v)
200 µl
Add all ingredients into a 500-ml Erlenmeyer flask. Swirl to disperse. Heat in a microwave oven on low or on a heating
stirrer until the agarose is completely dissolved. Do not allow the solution to boil over. Dispense 1.5-ml aliquots into
screw-cap tubes and store at room temperature.
132 80-6429-60 AD
Appendix II
Optimized silver staining of large-format DALT gels
and DALT 12.5 precast gels using PlusOne Silver
Staining Kit, Protein
Prepare staining reagents (250 ml per gel) according to the PlusOne Silver Staining Kit, Protein instructions with the
following exceptions:
1. Prepare twice the volume of fixing solution as indicated in the kit instructions (i.e. 500 ml per gel rather than 250 ml).
2. Prepare the developing solution with twice the volume of formaldehyde solution as indicated in the kit instructions
(i.e. 100 µl per 250 ml rather than 50 µl per 250 ml).
3. Stain the gels according to the following protocol:
Step Solutions
Amount
Time
Fixation
Ethanol
Acetic acid, glacial
Make up to 500 ml with distilled water
200 ml
50 ml
2 × 60* min
Sensitizing
Ethanol
Glutardialdehyde† (25% w/v)
Sodium thiosulfate (5% w/v)
Sodium acetate (17 g)
Make up to 250 ml with distilled water
75 ml
1.25 ml
10 ml
1 packet
60 min
Washing
Distilled water
5 × 8 min
Silver reaction
Silver nitrate solution (2.5% w/v)
Formaldehyde† (37% w/v)
Make up to 250 ml with distilled water
60 min
25 ml
0.1 ml
Washing
Distilled water
4 × 1 min
Developing
1 packet
100 µl‡
5 min¶
Stop
Sodium carbonate (6.25 g)
Formaldehyde (37%)
Make up to 250 ml with distilled water
Stir vigorously to dissolve sodium carbonate
Na EDTA.H O (3.65 g)
1 packet
45 min
2
2
Make up to 250 ml with distilled water
Washing
Distilled water
Preservation†
Glycerol (supplied at 87%, final conc. 4%)
11.5 ml**
20 min
Made up to 250 ml with distilled water
OR
25 ml
Ethanol (10% v/v)††
Made up to 250 ml with distilled water
2 × 30 min
* The first fixation may be prolonged up to 3 days if desired.
†
By omitting glutardialdehyde from the sensitizer and formaldehyde from the silver nitrate solution, as well as omitting the
“preservative step”, the method becomes compatible with mass spectrometry analysis, although sensitivity is reduced. If
glutardialdehyde and formaldehyde are to be used, add them just before staining.
‡
The volume of the formaldehyde in the developing solution can be varied from 100 µl up to 250 µl, depending on the amount of
protein and the number of spots since formaldehyde is consumed in the developing reaction by proteins. Add the formaldehyde
directly before use.
¶
Approximate time; this step may be visually monitored. The gels should be transferred to stop solution when the spots have reached
the desired intensity and before the staining background becomes too dark.
** For gels cast on plastic supports, increase the amount of glycerol to 25 ml.
††
Short- and long-term storage of gels is possible in 10% ethanol rather than glycerol, if gels are not being dried down. Glycerol is
necessary only if planning to dry down gels. Storage in ethanol allows the gels to be compatible with spot picking/mass spectrometry.
80-6429-60 AD 133
134 80-6429-60 AD
Appendix III
Colloidal Coomassie staining procedure
This method has been modified from Neuhoff et al. (83).
5% Coomassie Blue G-250 stock
(5% Coomassie Blue G-250, 10 ml)
Amount
Coomassie Blue G-250
0.5 g
Double-distilled water
to 10 ml
Stir for a few minutes to disperse the Coomassie Blue G-250. The dye will not dissolve completely.
Colloidal Coomassie Blue G-250 dye stock solution
(10% ammonium sulfate, 1% (w/w) phosphoric acid, 0.1% Coomassie Blue G-250, 500 ml)
Amount
Ammonium sulfate (FW 132.1)
50 g
Phosphoric acid 85% (w/w)
6 ml
5% Coomassie Blue G-250 stock
10 ml
Double-distilled water
to 500 ml
Colloidal Coomassie Blue G-250 working solution
(8% ammonium sulfate, 0.8% phosphoric acid, 0.08% Coomassie Blue G-250, 20% methanol, 500 ml)
Amount
Colloidal Coomassie Blue G-250
dye stock solution
400 ml
Methanol
100 ml
Prepare colloidal stain immediately before staining the gel.
1. Fix gel for at least 30 min in 10% acetic acid, 40% ethanol.
2. Decant the fixer and place the gel in colloidal stain (100–300 ml per gel, depending on size).
3. Leave overnight or longer. The staining gets more and more intense for up to 7 days.
3. Rinse gel repeatedly with water to remove residual stain.
4. Soak in 5% glycerol, 20% ethanol for no more than 30 min prior to drying.
The above step is necessary only if drying down the gel.
Ethanol will tend to shrink the gel and make it easier to handle, but it will also destain the gel.
80-6429-60 AD 135
136 80-6429-60 AD
Appendix IV
Protocol for use of Deep Purple Total Protein Stain
Reagents supplied in the kit
Deep Purple Total Protein Stain in 50% (v/v) DMSO and 50% (v/v) acetonitrile
Required but not provided
SDS (e.g. PlusOne code number 17-1313-01)
Acrylamide gel and other related electrophoresis reagents
Citric acid
Boric acid
Sodium hydroxide
High purity water (double distilled, RO or equivalent)
Ethanol
Ammonium carbonate
Acetonitrile
High-purity water (RO quality or better) should be used as a diluent for Deep Purple Total Protein Stain and for preparing all gel processing solutions.
All reagents used should be of the highest quality available since any impurities can affect the background
obtained on imaging. PlusOne reagents from GE Healthcare are recommended.
Critical parameters
Several critical parameters are important to the success of the Deep Purple Total Protein Stain protocol. Review these
parameters prior to beginning the procedure.
• Ensure that the containers used for gels are clean and do not contain any contaminants. A wide variety of
non-metallic containers can be used with this stain, including polypropylene, polystyrene, or Pyrex™ glass.
• Ensure that plates to be coated with Bind-Silane are prepared to the highest standard.
• Use gloves that are not powdered. Wash new gloves prior to handling plates, containers, or gels. Any powder
transferred to the gel may show up as speckles on images.
• During preparation of plates for gel casting, employ methods that minimize generation of dust particles. The use
of any type of paper towel will generate particulate matter that will be visualized as “speckles.” Plates should be
cleaned using lint-free cloths, such as Crew™ Wipes.
• During the protein staining step, a volume of working stain solution equivalent to at least a 10-fold excess of the gel
volume should be used. During all other steps a volume equivalent to ~20-fold excess of the gel volume should be
used (Table A).
• Do not dilute the stain beyond 1:200 as this will result in reduced intensity and sensitivity.
• Do not re-use the stain solution as this may result in a significant loss of sensitivity.
• During the process, gel containers should be covered to exclude light and agitated gently on a mixer platform.
• The source of SDS used to prepare and run polyacrylamide gels can affect the background obtained on imaging.
Use high-quality materials. Certain commercially available premade running buffers may not be suitable,
particularly when using short fixation times.
80-6429-60 AD 137
Table A. Typical stain and gel processing solution volumes for the Deep Purple Total Protein Stain protocol.
Electrophoresis system
Gel dimensions (cm)
Stain volume (ml)
Processing solution volume (ml)
Ettan DALTsix 20 × 26 × 0.1 500
1000
Ettan DALTtwelve 20 × 26 × 0.1 500
1000
miniVE
10 × 10 × 0.05 50
100
SE 260 10 × 10 × 0.05 50 100
SE 600 18 × 16 × 0.1 250
500
For optimal performance, it is critical that the pH in the gel is raised before adding the stain solution. If
not, high background or negative staining may be observed. Therefore, the proper wash solution in the
appropriate volumes must be used
Do not dilute the stain beyond 1:200 for gels and 1:400 for blots as this will result in reduced intensity and
sensitivity.
Do not reuse the stain solution as this may result in a significant loss of sensitivity.
If the whole staining process takes more than 8 hours; process gel containers should be covered to exclude light and
agitated gently on a mixer platform.
Solution and reagents required:
A. Fixation/Acidification solution for gels
15% (v/v) ethanol / 1% citric acid (v/v) in water (approx. pH 2.3). Add 150 ml of 99.9% ethanol and 10 g citric acid to
850 ml water (check pH). Fixing solution can be stored at room temperature for up to six months.
Note: Ethanol 15% (v/v) in water can be replaced by methanol 30% (v/v) in water. Citric acid 1% (v/v) in water can be
replaced by acetic acid 7.5% (v/v) in water. The concentration of acetic acid should not exceed 10%.
B. Working stain solution for gels and blots
100 mM sodium borate, pH 10.5 in water. Dissolve 6.2 g boric acid in 800 ml water and adjust pH to 10.5 with NaOH,
then make to 1 l. This solution should be made fresh at the time of use by adding 1 part Deep Purple to 200 parts
borate buffer for gels or 1 part Deep Purple to 400 parts of borate buffer for blots. Boric acid/NaOH buffer may be
prepared in advance and can be stored for up to 6 months.
C. Wash solution for gels
15% (v/v) ethanol in water. Add 150 ml ethanol to 850 ml water. Washing solution may be prepared in advance and
can be stored at room temperature for up to 6 months.
Note: Ethanol 15% (v/v) in water can be replaced by methanol 30% (v/v) in water. We recommend that you
consistently use either methanol or ethanol throughout the procedure.
D. Storage solution for gels
Gels should be stored at 4ºC protected from light in 1% citric acid. Add 10 g citric acid to 1 l water (pH 2.3). For
extended storage (up to 6 months), add Deep Purple (1:200) to the storage solution.
Protocol
Low-fluorescence glass plates should be used for plastic-backed gels, as these backed gels have problems
with background.
Gel electrophoresis
Perform electrophoresis according to established techniques.
Note: If visual orientation is required on 1D gels, Rainbow™ Markers (RPN800) may be used. If a tracking dye is used in
the loading buffer, such as bromophenol blue, it is recommended to run the dye front just off the bottom of the gel.
138 80-6429-60 AD
Fixation
1. Place an appropriate volume of 15% ethanol /1% citric acid (v/v, solution A) into the containers that will be used to process gels. The recommended volume of fixation solution required is ~20 fold excess of the gel volume (Table A).
Note: Alternative fixation solutions that have been used successfully with Deep Purple Total Protein Stain are:
• 7.5% acetic acid/10% ethanol
• 7.0% acetic acid/30% ethanol
2. Dismantle the electrophoresis apparatus.
For free-floating gels, remove the gel from the plates by floating the gel off with gentle agitation in the fix
solution.
For backed gels, place the gel and plate directly into the fix solution.
Note: Place only one gel in each container. The stacking gel can be left attached to help with gel orientation.
3. Incubate in the fixation solution A, for a minimum of 1 hour, at room temperature with gentle agitation.
Note: Overnight fixation should be used for backed gels, large format gels and thick gels (> 1.5 mm) and it is also
recommended for applications where maximum sensitivity is required.
Staining
1. Take the stain out of the freezer (-15 to -30 °C) and allow to stand at room temperature for 15-30 min. Prepare
working stain solution B.
2. Pour off the fixation solution and replace with working stain solution B in ~20-fold excess.
Try to minimize carry-over of the acidic fixation solution. Stain for 1 hour (1.0 mm thick gels). For gels 1.5 mm thick or
backed gels, increase the staining time to 1.5 hours. Extending the staining time up to 2 hours will not adversely
affect results. There will be some loss in fluorescence intensity if the staining time is greater than 3 hours.
Note: The staining solution degrades over time and should not be stored. If the total staining procedure is extended
to more than eight hours, containers can be wrapped in foil or covered with black plastic. Alternatively, containers
and lids of a solid colored plastic may be used.
3. Pour off the stain solution and wash the gels by gentle rocking in wash solution C for 30 minutes. This step should be
increased to 45 min for 1.5 mm gels or if you experience high background fluorescence.
4. Remove gels from wash solution and acidify by placing them in solution A and rock gently for 30 min. This step may
be repeated or extended up to overnight to reduce background staining. If this step is prolonged to over night, gels
should be protected from light. Prior to imaging, gels should be rinsed 5 minutes in wash solution C. The gel can be
imaged at this stage.
Note: If the gels swell during the staining process, soak the gels in solution A for 30 min prior to analysis.
After imaging, the gels can be stored at 4ºC protected from light in gel 1% citric acid (solution D). For longterm
storage (more than 6 months), add Deep Purple (1:200 dilution) to the storage solution. If stored, gels should be rinsed
(2 x 15 min) in 15% ethanol (solution C) prior to imaging. Acidifying in 15% ethanol/1% citric acid (solution A) for 15
minutes may be used to reduce background.
Visualization
A. Flat-bed laser-based fluorescence imaging systems
1. Ensure that the scanning bed of the laser is clean and free from smears and particles. Follow recommended
procedures provided with the instrument.
Note: On the Typhoon scanner it has been shown that fluorescent contamination on the platen can be eliminated
by wiping the surface with 10% (v/v) H2O2 (hydrogen peroxide) followed by a rinse with double-distilled water
(see the user manual for full details).
2. Set up the scanner as recommended in the relevant system operational manual. For example, the following
settings are recommended for use with a Typhoon scanner:
Excitation: Green laser (532 nm)
Emission: 560LP or 610BP filter.
Pre-scan using 1000 micron resolution and then scan using a 100 micron resolution.
Note: If the pre-scan shows saturated bands/spots, reduce the PMT voltage rating and pre-scan again. If the
pre-scan shows too low signal increase the PMT voltage rating and pre-scan again. Deep Purple Total Protein
Stain can also be imaged on a Typhoon scanner using the blue laser (457 nm or 488 nm). If using an alternate
fluorescent scanner, for the best optimal images, scan using as similar settings as possible to those recommended.
80-6429-60 AD 139
3. Process the image according to experimental requirements and the instructions for the relevant software program.
B.
1.
Imaging with UV light sources
Place the gel onto the UV transilluminator (302 or 365 nm wavelength emission required) and follow the operating
and safety instructions as relevant for the excitation instrument and imaging system. Images can be captured
using appropriate camera systems and filters (film, video, CCD).
Note: For long periods of illumination it is advisable to place the gel on a glass plate, raised on spacers above the
transilluminator, in order to reduce heat damage to the stained proteins. Cooling the gel prior to visualization can
also help reduce degradation.
2. If required, pick any bands/spots.
Note: If manually picking bands/spots, it is advisable to place gels on a glass plate in order to reduce possible
damage to the instrument surface. Prolonged, continuous exposure to a strong UV light source will degrade the
Deep Purple Total Protein Stain signal, with a half-life in the region of 15 to 30 min.
Refer to the Deep Purple Total Protein Stain product instructions for additional information on re-staining of gels,
alternative staining trays, alternative imaging instruments, use of Deep Purple Total Protein Stain with Ettan DIGE, and
cleaning of imaging instruments. For cleaning and preparation of Bind-Silane coated plates, refer to appendix V.
The instructions accompanying Deep Purple Total Protein Stain also include a troubleshooting guide.
140 80-6429-60 AD
Appendix V
Treating glass plates with Bind-Silane
Spot picking with Ettan Spot Picker or Spot Handling Workstation requires that gels are precast on backing (e.g. Ettan
DALT II Precast Gel 12.5) or immobilized on backing during casting. Two different types of backing may be used: GelBond PAGfilm or a glass plate, treated with Bind-Silane solution.
To scan a gel with fluorescently labeled proteins, it is important that GelBond not be used for the gel backing.
GelBond is a plastic material and fluoresces intensely at the wavelengths used for scanning.
Protocol to treat glass plates with Bind-Silane
The glass plates must be properly cleaned. Before re-use, soak the plates overnight in a 5% Decon™ 90 solution. Do not leave plates standing in a Decon solution for a longer time as this will eventually cause etching
due to the alkali nature of Decon.
1. Thoroughly wash each plate to be treated. Take care to remove any gel fragments attached to the plate from
previous gels. The careful cleaning of the glass plates before casting is important, to ensure a uniform coating
with the Bind-Silane and to avoid keratin contamination.
2. Thoroughly rinse the plates with double-distilled water to remove Decon.
3. Dry the plates using a lint-free tissue or leave them to air dry.
4. Prepare the Bind-Silane working solution:
Ethanol
8 ml
Glacial acetic acid
200 µl
Bind-Silane
10 µl
Double-distilled water
1.8 ml
5. Pipette 2–4 ml (depending on plate size) of the Bind-Silane working solution onto each plate and distribute equally
over the plate with a lint-free tissue such as Crew Wipes.
Cover the plate to prevent dust contamination and leave to air dry on the bench for 1–1.5 h.
6. Polish the plate with a lint-free tissue such as Crew Wipes, moistened with a small amount of double-distilled
water or ethanol.
The gels will stay attached to the glass during electrophoresis, staining procedures, scanning, and storage.
80-6429-60 AD 141
142 80-6429-60 AD
Appendix VI
Using Ready-Sol
PlusOne ReadySol IEF, 40%T and 3%C (see ordering information) is a premade stock solution of acrylamide and
bisacrylamide. Note that the %C will not change on dilution as it is a ratio. The table below provides the recipe for
making 100 ml of each percentage gel.
5% 7.5%
10%
12.5%
15%
Monomer stock solution (ReadySol 40%T, 3% C) (ml)
12.5
18.75
25.0
31.25
37.5
4× Resolving gel buffer (ml)*
25.0
25.0
25.0
25.0
25.0
10% SDS (ml)
1.0
1.0
1.0
1.0
1.0
Double-distilled water (ml) 61.0
54.75
48.5
42.25
36.0
10% Ammonium persulfate† (ml)
TEMED†
0.5
0.5
0.5
0.5
0.5
33 µl
33 µl
33 µl
33 µl
33 µl
* 4× Resolving gel buffer is 1.5 M Tris base, ph 8.8. To make, mix 181.7 g Tris base with 750 ml double-distilled water, adjust the pH to
8.8 with HCl, and make up to final volume of 1 l with double-distilled water. See also solution H in appendix I.
†
Add ammonium persulfate and TEMED just before casting the gel.
80-6429-60 AD 143
144 80-6429-60 AD
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Electrophoresis 19, 845–851 (1998).
64. Wildgruber, W. et al. Web-based two-dimensional database of Saccharomyces cerevisiae proteins using immobilized pH gradients
from pH 6 to pH 12 and matrix-assisted laser desorption/ionization-time of flight mass spectrometry. Proteomics 2, 727–732 (2002).
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Weinheim (2002).
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148 80-6429-60 AD
Additional reading and reference material
Item (literature type in parentheses)
Code No.
2-D electrophoresis: a comparison of carrier ampholyte
and immobilized pH gradients (scientific poster)
80-6419-53
Automated staining of polyacrylamide gels with Hoefer Processor Plus
(technical manual)
80-6343-34
Blot processing with the Processor Plus (handbook)
80-6447-27
Comparison of Deep Purple Total Protein Stain and Sypro Ruby in 1-D and 2-D gel electrophoresis (application note)
18-1177-44
CyDye DIGE Fluors and Labeling Kits (data file)
18-1164-84
DeCyder 2-D Differential Analysis Software v 7.0 (data file)
28-4015-43
DIGE Gel and DIGE Buffer Kit (data file)
28-9480-26
Detection and mass spectrometry identification of protein changes in low-abundance tissue using CyDye DIGE Fluor saturation dyes (data file)
18-1177-51
Electrophoresis in Practice
Westermeier, R., Wiley-VCH Verlag GmbH, Weinheim (2001)
18-1124-59
DALTsix Large Vertical Electrophoresis System for second
dimension 2-D electrophoresis (data file)
80-6490-02
Fluorescence Imaging: principles and methods (handbook)
63-0035-28
ImageMaster 2-D Platinum (data file)
18-1177-20
Immobiline DryStrip Gels (data file)
18-1177-60
Immobiline DryStrip visualization of pH gradients (technical brochure)
18-1140-60
Immobiline DryStrip (instructions)
28-9537-55
Improved detection and identification of low-abundance human bronchoalveolar lavage fluid (BALF) proteins using 2-D electrophoresis and
Ettan MALDI-ToF mass spectrometry (application note)
18-1151-35
Improved spot resolution and detection of proteins in 2-D electrophoresis using 24 cm Immobiline DryStrip gels (application note)
18-1150-23
Multiple mini-format 2-D electrophoresis using Hoefer™ SE 600 Standard Vertical Electrophoresis Unit (application note)
80-6445-94
Multiple mini-format 2-D electrophoresis. Multiphor II Flatbed
(application note)
80-6443-47
PlusOne sample preparation kits and reagents (brochure)
80-6487-74
Protein analysis - using the power of 2-D electrophoresis (brochure)
18-1124-82
Protein electrophoresis (technical manual)
80-6013-88
Proteomics in Practice, A Laboratory Manual of Proteome Analysis,
Westermeier, R. and Naven, T., Wiley-VCH Verlag GmbH, Weinheim (2002)
18-1164-45
Typhoon Variable Mode Imager (data file)
63-0048-48
User manuals
Ettan DIGE
18-1164-40
DeCyder 2D™ 7.0 User Manual
28-9414-47
Ettan DALTsix
80-6492-49
Ettan Ettan Gel 12.5 and Ettan DALT Buffer Kit
71-5019-56
Ettan IPGphor 3
11-0034-58
miniVE
80-6420-86
Multiphor II Electrophoresis System
18-1103-43
SE 260 80-6291-95
SE 600 Ruby 80-6353-79
Many of these items can be downloaded from the Literature section on www.gehealthcare.com
80-6429-60 AD 149
150 80-6429-60 AD
Ordering information
Product
Quantity
Code No.
Sample Grinding Kit
50 samples
80-6483-37
Protease Inhibitor Mix
1 ml
80-6501-23
illustra triplePrep Kit
50 preps
28-9425-44
Nuclease Mix
0.5 ml
80-6501-42
Albumin and IgG Removal Kit
10 samples
RPN6300
2-D Clean-Up Kit
50 samples
80-6484-51
2-D Quant Kit
500 assays
80-6483-56
2-D Protein Extraction Buffer Trial Kit 6 x 10 ml
28-9435-22
Mini Dialysis Kit, 1 kDa cut-off, up to 250 µl
50 samples
80-6483-75
Mini Dialysis Kit, 1 kDa cut-off, up to 2 ml
50 samples
80-6483-94
Mini Dialysis Kit, 8 kDa cut-off, up to 250 µl
50 samples
80-6484-13
Mini Dialysis Kit, 8 kDa cut-off, up to 2 ml
50 samples
80-6484-32
Vivaspin 6 MWCO 50 000
25 pack
28-9323-18
Sample preparation kits and reagents
Spectrophotometer
Ultrospec 3100 pro UV/Visible Spectrophotometer
Inquire
First-dimension products and accessories
Ettan IPGphor 3 Isoelectric Focusing System
Ettan IPGphor 3 Isoelectric Focusing Unit
1
11-0033-64
Ettan IPGphor Manifold, Complete
80-6498-38
IPGbox kit
1
28-9334-92
Sample cups
120/pk
80-6498-95
Paper electrode
240/pk
80-6499-14
Paper bridge pads
120/pk
80-6499-33
Multiphor II Electrophoresis System and accessories
Multiphor II Electrophoresis Unit
18-1018-06
MultiTemp III Thermostatic Circulator, 115 V
18-1102-77
MultiTemp III Thermostatic Circulator, 230 V
18-1102-78
EPS 3501 XL Power Supply
18-1130-05
Immobiline DryStrip Kit and other accessories
Immobiline DryStrip Kit (for use with Multiphor II only)
18-1004-30
Immobiline DryStrip Cover Fluid
1 l
17-1335-01
Sample cups 60/pk
18-1004-35
IEF electrode strips
100/pk
18-1004-40
See also under Second-dimension products and accessories
Strip Holders for use with Immobiline DryStrip and Ettan IPGphor 3 Isoelectric Focusing System
7 cm
1/pk
6/pk
80-6416-87
80-6416-11
11 cm
1/pk
6/pk
80-6417-06
80-6416-30
13 cm
1/pk
6/pk
80-6417-25
80-6416-49
18 cm
1/pk
6/pk
80-6417-44
80-6416-68
24 cm
1/pk
6/pk
80-6470-07
80-6469-88
Immobiline DryStrip Cover Fluid
1 l
17-1335-01
Cleaning Solution, Strip Holder 950 ml
80-6452-78
80-6429-60 AD 151
Product
Quantity
Code No.
Immobiline DryStrip gels (all 12/pk)
7 cm
pH 3–5.6 NL
pH 3–10
pH 3–10 NL
pH 3–11 NL
pH 5.3–6.5
pH 6.2–7.5
pH 4–7
pH 6–11
pH 7–11 NL
17-6003-53
17-6001-11
17-6001-12
17-6003-73
17-6003-58
17-6003-63
17-6001-10
17-6001-94
17-6003-68
11 cm
pH 3–5.6 NL
pH 3–10
pH 3–11 NL
pH 4–7
pH 5.3–6.5
pH 6–11
pH 6.2–7.5
pH 7–11 NL
17-6003-54
18-1016-61
17-6003-74
18-1016-60
17-6003-59
17-6001-95
17-6003-64
17-6003-69
13 cm
pH 3–5.6 NL
pH 3–10
pH 3–10 NL
pH 3–11 NL
pH 4–7
pH 5.3–6.5
pH 6–11
pH 6.2–7.5
pH 7–11 NL
17-6003-55
17-6001-14
17-6001-15
17-6003-75
17-6001-13
17-6003-60
17-6001-96
17-6003-65
17-6003-70
18 cm
pH 3–5.6 NL
pH 3–10
pH 3–10 NL
pH 3–11 NL
pH 4–7
pH 6–11
pH 6–9
pH 6.2–7.5
pH 7–11 NL
17-6003-56
17-1234-01
17-1235-01
17-6003-76
17-1233-01
17-6001-97
17-6001-88
17-6003-66
17-6003-71
24 cm
pH 3–5.6 NL
pH 3–10
pH 3–10 NL
pH 3–11 NL
pH 3.5-4.5
pH 4–7
pH 6–9
pH 3–7 NL
pH 6.2–7.5
pH 7–11 NL
17-6003-57
17-6002-44
17-6002-45
17-6003-77
17-6002-38
17-6002-46
17-6002-47
17-6002-43
17-6003-67
17-6003-72
Equilibration Tube Set for up to
24 cm IPG strips
152 80-6429-60 AD
12/pk
80-6467-79
Product
Quantity
Code No.
DeStreak Rehydration Solution
5 × 3 ml
17-6003-19
DeStreak Reagent
1 ml
17-6003-18
DeStreak rehydration reagents
IPG Buffer, 1 ml
pH 3.5–5.0
17-6002-02
pH 5.5–6.7
17-6002-06
pH 4–7 17-6000-86
pH 6–11
17-6001-78
pH 3–10
17-6000-87
pH 3–10 NL
17-6000-88
pH 7–11 NL 17-6004-39
pH 3–11 NL 17-6004-40
Pharmalyte, 25 ml
pH 3–10
17-0456-01
pH 5–8
17-0453-01
pH 8–10.5
17-0455-01
Second-dimension products and accessories
Mini-Vertical units and accessories
miniVE complete, includes 3 rectangular glass plates, 3 notched plates, 2 gel modules, lid, lower buffer chamber, 2 each 1.0-mm thick
10 well combs and 1.0-mm thick spacer sets (glass plate size: 10 × 10.5 cm)
80-6418-77
Spacer, 1.0 mm
2/pk
80-6150-11
Spacer, 1.5 mm
2/pk
80-6150-30
SE 250 Mini-Vertical Unit,
complete, for 2 slab gels (gel format 10 × 8 cm)
80-6147-45
SE 260 Mini II Vertical Unit,
complete, for 2 slab gels (gel format 10 × 10.5 cm)
80-6149-35
SE 235 Mighty Small 4-Gel Caster, complete
80-6146-12
SE 245 Mighty Small Dual Gel Caster
80-6146-50
Wonder Wedge plate separation tool
80-6127-88
SE 600 Ruby Vertical Electrophoresis System and accessories
SE 600 Ruby Dual Cooled Vertical Gel Unit for
up to four gels (glass plate size: 18 × 16 cm)
80-6479-57
Spacer, 1.0 mm, 1 cm wide 2/pk
80-6179-94
Spacer, 1.0 mm, 2 cm wide 2/pk
80-6180-70
Spacer, 1.5 mm, 1 cm wide 2/pk
80-6180-13
Spacer, 1.5 mm, 2 cm wide 2/pk
80-6180-89
SE 615 Multiple Gel Caster for 2 to 10 gels (glass plate size: 18 × 16 cm)
80-6182-79
Glass plates, 18 × 8 cm 2/pk
80-6186-59
Glass plates, 18 × 8 cm low fluorescence
2/pk
80-6475-77
Divider glass plate, 18 × 8 cm, notched
80-6186-78
Glass plates, 18 × 16 cm 2/pk
80-6178-99
Glass plates, 18 x 16 cm low fluorescence
2/pk
80-6442-14
Divider glass plate, 18 × 16 cm, notched
80-6179-18
Clamp assembly, 8 cm
2/pk
80-6187-35
Clamp assembly, 16 cm
2/pk
80-6173-29
80-6429-60 AD 153
Product
Quantity
Code No.
Ettan DALT Large Vertical Systems and accessories
Ettan DALTtwelve Separation Unit and
Power Supply/Control Unit, 115 VAC
80-6466-46
Ettan DALTtwelve Separation Unit and
Power Supply/Control Unit, 230 VAC
80-6466-27
Ettan DALTsix Separation Unit and
Power Supply/Control Unit, 115 VAC
80-6485-08
Ettan DALTsix Separation Unit and
Power Supply/Control Unit, 230 VAC
80-6485-27
Ettan DALTtwelve Gel Caster, complete, includes 5 filler and 16 separator sheets (order cassettes separately)
80-6467-22
Ettan DALTsix Gel Caster, complete, includes 6 filler and 7 separator sheets (order cassettes separately)
80-6485-46
Ettan DALT Cassette Removal Tool 2/pk
80-6474-82
Ettan DALT Buffer Seal Removal Tool 2/pk
80-6474-63
Ettan DALT Precast Gel Cassette
80-6466-65
Ettan DALT Gel Casting Cassette, 1.0 mm (hinged cassette)
80-6466-84
Ettan DALT Gel Casting Cassette, 1.5 mm (hinged cassette)
80-6488-69
Ettan DALT Blank Cassette Insert
80-6467-03
Roller (for precast gels)
80-1106-79
Wonder Wedge plate separation tool (for lab-cast gels)
80-6127-88
Ettan DALT Separator Sheets 0.5 mm
16/pk
80-6467-41
Ettan DALT Filler Sheets 1.0 mm
6/pk
80-6467-60
Ettan DALT Cassette Rack
2/pk
80-6467-98
Ettan DALT Glass Plate Set, including spacers
(standard glass plates for spot picking)
1 set of 2 pcs
80-6475-39
Ettan DALT Low Fluorescence Glass Plate Set, including spacers 1 set of 2 pcs
80-6475-58
Equilibration Tube Set
12
80-6467-79
Staining Tray Set
80-6468-17
Ettan DALTsix Gradient Maker
80-6487-36
DALT Gradient Maker with peristaltic pump, 115 V
80-6067-65
DALT Gradient Maker with peristaltic pump, 230 V
80-6067-84
DIGE gels and buffer kit
DIGE gel
3
28-9374-51
DIGE Buffer Kit
28-9374-52
2 × 125 ml anode buffer
4 × 125 ml cathode buffer
12 tubes agarose sealing solution
Ettan DALT precast gels and buffer kit
DALT Gel 12.5
6/pk
DALT Buffer Kit
17-6002-36
17-6002-50
Gradient makers
SG15 Gradient Maker, 15 ml total volume
SG 30 Gradient Maker, 30 ml total volume
SG 50 Gradient Maker, 50 ml total volume
SG 100 Gradient Maker, 100 ml total volume
SG 500 Gradient Maker, 500 ml total volume
80-6197-61
80-6197-80
80-6197-99
80-6196-09
80-6198-18
Multiphor II Electrophoresis System
Multiphor II Electrophoresis Unit
Multiphor II Buffer Strip Positioner
IEF sample application pieces
200/pk
18-1018-06
80-6442-90
80-1129-46
154 80-6429-60 AD
Product
Quantity
Code No.
Power supplies
EPS 3501 XL Power Supply, 3500 V, 400 mA, 200 W
18-1130-05
EPS 2A200 Power Supply, 200 V, 2000 mA, 200 W 80-6406-99
EPS 301 Power Supply, 300 V, 400 mA, 80 W
18-1130-01
EPS 601 Power Supply, 600 V, 400 mA, 100 W
18-1130-02
EPS 1001 Power Supply, 1000 V, 400 mA, 100 W
18-1130-03
Thermostatic circulator
MultiTemp III Thermostatic Circulator, 115 V
18-1102-77
MultiTemp III Thermostatic Circulator, 230 V
18-1102-78
ExcelGel SDS gels
ExcelGel SDS 2-D Homogeneous 12.5
6/pk
17-6002-21
ExcelGel SDS Gradient XL 12–14
3/pk
17-1236-01
ExcelGel SDS Buffer Strips, anode and cathode
6 each/pk
17-1342-01
Acrylamide PAGE (acrylic acid < 0.05%)
250 g
17-1302-01
Acrylamide PAGE (acrylic acid < 0.05%)
1 kg
17-1302-02
Acrylamide IEF (acrylic acid < 0.002%)
250 g
17-1300-01
Acrylamide IEF (acrylic acid < 0.002%)
1 kg
17-1300-02
Acrylamide IEF, 40% solution
1 l
17-1301-01
Acrylamide PAGE, 40% solution
1 l
17-1303-01
N,N’-methylenebisacrylamide
25 g
17-1304-01
N,N’-methylenebisacrylamide
100 g
17-1304-02
N,N’-methylenebisacrylamide, 2% solution
1 l
17-1306-01
ReadySol IEF, 40% T and 3% C
1 l
17-1310-01
Agarose NA
10 g
17-0554-01
Glycine
500 g
17-1323-01
Ammonium persulfate
25 g
17-1311-01
TEMED
25 ml
17-1312-01
Glycerol, 87%
1 l
17-1325-01
SDS
100 g
17-1313-01
Thiourea
100 g
RPN6301
Iodoacetamide
25 g
RPN6302
Tris
500 g
17-1321-01
Urea 500 g
17-1319-01
CHAPS
1 g
17-1314-01
Triton X-100 500 ml
17-1315-01
Dithiothreitol (DTT) 1 g
17-1318-01
Bromophenol Blue
10 g
17-1329-01
Bind-Silane
25 ml
17-1330-01
Immobiline DryStrip Cover Fluid
1 l
17-1335-01
Amberlite IRN-150L
500 g
17-1326-01
Nuclease Mix
0.5 ml
80-6501-42
Deoxyribonuclease I (DNase I)
20 mg
27-0516-01
Ribonuclease I (RNase A and RNase B)
1 g
27-0330-02
Ribonuclease I “A” (RNase A)
100 mg
27-0323-01
PlusOne chemicals and reagents
Enzymes
80-6429-60 AD 155
Product
Quantity
Code No.
Molecular weight markers
Peptide Marker Kit (Mr range 2512–16 949)
80-1129-83
LMW-SDS Marker Kit (Mr range 14 400–97 000)
17-0446-01
HMW-SDS Marker Kit (Mr range 53 000–220 000)
17-0615-01
Full-Range Rainbow Molecular Weight Markers
RPN800
pI calibration kit
Carbamylyte Calibration Kit
17-0582-01
Automated gel and blot processing
Processor Plus Base Unit
(includes Base Unit, Reagent Tubing, and Protocol Key)
80-6444-04
Accessories to make functional for staining and/or blotting:
Staining Tray Pack, 19 × 29 cm
(complete with gel staining tray base, tray, and lid)
80-6444-80
Staining Tray Pack, 29 × 35 cm
(includes gel staining tray base, tray, and lid)
80-6445-18
Blot Processing Tray Pack
80-6444-23
(includes tray base, disposable mini and standard trays, lid,
reagent bottles and rack, and vented lid for waste products)
Additional accessories for Processor Plus
Stainless Steel Staining Tray 19 × 29 cm 80-6343-91
Stainless Steel Staining Tray 29 × 35 cm
80-6345-24
Blot Processing Mini Tray
3/pk
80-6444-42
Blot Processing Standard Tray
3/pk
80-6444-61
Manual gel staining
Stainless Steel Staining Tray Set
80-6468-17
Staining reagents
Silver Staining Kit, Protein
17-1150-01
Coomassie tablets, PhastGel Blue R-350
17-0518-01
Deep Purple Total Protein Stain
(sufficient for two large-format gels or 20 minigels)
RPN6305
Deep Purple Total Protein Stain
(sufficient for 10 large-format gels or 100 minigels)
RPN6306
Gel dryers
GD 2000 Vacuum Gel Dryer for gels up to 33 × 44 cm, 115 V
80-6428-84
GD 2000 Vacuum Gel Dryer for gels up to 33 × 44 cm, 230 V
80-6429-03
Cellophane Sheets
80-6117-81
Image analysis systems and software
Image Scanner III
28-9076-07
Typhoon FLA 9000
28-9558-08
ImageMaster 2D Platinum
18-1176-30
ImageMaster 2D Platinum site license
18-1176-31
See also under 2-D DIGE products
156 80-6429-60 AD
Product
Quantity
Code No.
Spot handling
Ettan Spot Picker
18-1145-28
Ettan Digester
18-1142-68
2-D DIGE products
CyDye DIGE Fluor Minimal Dye Labeling Kit
25-8010-65
(includes Cy2, Cy3, and Cy5) (5nmol)
CyDye DIGE Fluor Cy2 minimal dye, 5 nmol
25-8010-82
CyDye DIGE Fluor Cy2 minimal dye, 10 nmol
25-8008-60
CyDye DIGE Fluor Cy2 minimal dye, 25 nmol
RPK0272
CyDye DIGE Fluor Cy3 minimal dye, 5 nmol
25-8010-83
CyDye DIGE Fluor Cy3 minimal dye, 10 nmol
25-8008-61
CyDye DIGE Fluor Cy3 minimal dye, 25 nmol
RPK0273
CyDye DIGE Fluor Cy5 minimal dye, 5 nmol
25-8010-85
CyDye DIGE Fluor Cy5 minimal dye, 10 nmol
25-8008-62
CyDye DIGE Fluor Cy5 minimal dye, 25 nmol
RPK0275
CyDye DIGE Fluor Labeling Kit for Scarce Samples
(for a minimum of 12 labeling reactions)
25-8009-83
CyDye DIGE Fluor Labeling Kit for Scarce Samples
plus Preparative Gel Labeling
(for minimum of 12 labeling reactions and 1 prep gel)
25-8009-84
CyDye DIGE Fluor Preparative Gel Labeling Kit for Scarce Samples
28-9366-83
CyDye DIGE Kit, 2 nmol
28-9345-30
DIGE Trial pack: CyDye DIGE Kit, 2 nmol + DeCyder 1-month trial license
28-9373-73
Ettan DIGE Gel Alignment Guides for SE600
80-6496-29
Ettan DIGE Gel Alignment Guides for Ettan DALT
80-6496-10
Imaging systems
Typhoon Variable Mode Imagers
Typhoon FLA 9000
1
28-9558-08
Typhoon FLA 7000
1
28-9558-09
Typhoon 9400
1
63-0055-78
Typhoon 9410
1
63-0055-80
Typhoon Trio
1
63-0055-87
Typhoon Trio+
1
63-0055-89
ImageQuant LAS 4000
1
28-9558-10
ImageQuant LAS 4010
1
28-9558-11
ImageQuant LAS 4000 mini
1
28-9558-13
ImageScanner III
1
28-9076-07
Storm 820 and ImageQuant TL
1
28-9328-12
Storm 845 and ImageQuant TL
1
28-9326-41
Storm 865 and ImageQuant TL
1
28-9327-91
ImageQuant™ imagers
Other imaging systems
80-6429-60 AD 157
Product
Quantity
Code No.
Analysis software
ImageQuant TL
ImageQuant TL, single user license
1
28-9236-62
ImageQuant TL, 5-user network license
1
28-9206-39
ImageQuant TL, 10-user network license
1
28-9236-57
ImageQuant TL 7.0 and ImageQuant TL SecurITy 8.0 Software
Package (with Getting Started Guide)
1
28-9380-94
ImageQuant TL 7.0 and ImageQuant TL SecurITy 8.0, single user license
1
28-9332-73
ImageMaster 2D Platinum
ImageMaster 2D Platinum 7.0 DIGE, 1 license
1
28-9380-55
ImageMaster 2D Platinum 7.0 upgrade to DIGE, 1 license
1
28-9398-10
ImageMaster 2D Platinum 7.0, 1 license
1
28-9380-91
ImageMaster 2D Platinum 6.0 upgrade to 7.0, 1 license
1
28-9399-70
ImageMaster 2D Platinum 7.0 software package
1
28-9408-30
DeCyder 2D
DeCyder 2D Oracle 10gR2, 5 user license
1
28-9435-88
DeCyder 2D 7.0, 1-user license
1
28-9442-75
DeCyder 2D 7.0, additional 1-user license
1
28-9442-77
DeCyder 2D 7.0, 1-user trial license
1
28-9442-79
DeCyder 2D 7.0, 1-user license (upgrade from 6.5 2D)
1
28-9442-80
DeCyder 2D 7.0, 1-user license (upgrade from 6.5 + EDA)
1
28-9442-81
DeCyder 2D 7.0, pre-installed computer 1-user license
(computer with DeCyder pre-installed,
Software Package, and e-license,
1 user node locked)
1
28-9435-86
DeCyder 2D 7.0 Software Package, (Installation Guide, Getting Started Guide,
and DeCyder 2D DVD case with installation
disc packaged in DeCyder box)
1
28-9435-83
DeCyder 2D 7.1 SPN, 1-user license
1
28-9763-18
DeCyder 2D 7.1 SPN, 1-user trial license
1
28-9763-20
DeCyder 2D 7.1 SPN, 1-user license (upgrade from 2D 7.0 )
1
28-9763-21
DeCyder 2D 7.1 SPN Software Package, (Installation Guide, Getting Started Guide,
and DeCyder 2D DVD case with installation
disc packaged in DeCyder box)
1
28-9757-78
158 80-6429-60 AD
Recommended additional consumables
Sulfobetaines
Calbiochem
PefaBloc
Merck
DMF (N,N’-dimethylformamide) 99.8% anhydrous
22,705-6
Sigma-Aldrich
Crew Wipes
Z23681-0
Sigma-Aldrich
L-lysine
L-5626
Sigma-Aldrich
Molecular Sieves 4Å
M2635
Sigma-Aldrich
Decon 90
cln 010
010 M.J. Patterson (Scientific) Ltd.
80-6429-60 AD 159
GE Healthcare
Cy, CyDye, DeCyder, Deep Purple, ECL, Ettan, ExcelGel, Hybond, illustra, ImageMaster,
ImageQuant, ImageScanner, Immobiline, IPGphor, Multiphor, MultiTemp, Personal Densitometer,
Pharmalyte, Rainbow, Scierra, and Typhoon are trademarks of GE Healthcare companies.
2-D Fluorescence Difference Gel Electrophoresis (2-D DIGE) technology is covered by US
patent numbers 6,043,025, 6,127,134 and 6,426,190 and equivalent patents and patent
applications in other countries and exclusively licensed from Carnegie Mellon University.
CyDye: this product or portions thereof is manufactured under an exclusive license from
Carnegie Mellon University under US patent numbers 5,569,587, 5,627,027 and equivalent
patents in other countries. The purchase of CyDye DIGE Fluors includes a limited license
to use the CyDye DIGE Fluors for internal research and development, but not for any
commercial purposes. A license to use the CyDye DIGE Fluors for commercial purposes is
subject to a separate license agreement with GE Healthcare.
CyDye: This product or portions thereof is manufactured under an exclusive license from
Carnegie Mellon University under US patent number 5,268,486 and equivalent patents in
the US and other countries. The purchase of CyDye products includes a limited license to
use the CyDye products for internal research and development but not for any commercial
purposes. A license to use the CyDye products for commercial purposes is subject to a
separate license agreement with GE Healthcare. Commercial use shall include:
1. Sale, lease, license or other transfer of the material or any material derived or
produced from it.
2. Sale, lease, license or other grant of rights to use this material or any material derived
or produced from it.
3. Use of this material to perform services for a fee for third parties, including contract
research and drug screening.
If you require a commercial license to use this material and do not have one, return this
material unopened to GE Healthcare Bio-Sciences AB, Bjorkgatan 30, SE-751 84 Uppsala,
Sweden and any money paid for the material will be refunded.
DeCyder: This release of DeCyder (software) is provided by GE Healthcare to the customer under
a nonexclusive license and is subject to terms and conditions set out in the 2-D Differential
Gel Electrophoresis Technology Access Agreement. Customer has no rights to copy or
duplicate or amend the Software without the prior written approval of GE Healthcare.
2-D Electrophoresis – Principles and Methods
GE, imagination at work, and GE monogram are trademarks of General Electric Company.
Deep Purple Total Protein Stain is exclusively licensed to GE Healthcare from
Fluorotechnics Pty Ltd.Deep Purple Total Protein Stain may only be used for applications
in life science research.Deep Purple is covered under a granted patent in New Zealand
entitled “Fluorescent Compounds”, patent number 522291 and equivalent patents and
patent applications in other countries.
DIGE Gel and DIGE Buffer Kit: The buffer system in this gel and buffer kit is covered by
patent application WO9616724 granted in US, EP and JP.
Ettan CAF MALDI Sequencing Kits are protected by patents owned by Procter & Gamble
Company and exclusively licensed to GE Healthcare Bio-Sciences AB and by joint patents
issued to both companies. The purchase of Ettan CAF MALDI Sequencing Kits includes a
limited license to use the technology for internal research and development, but not for
any commercial purposes. No right to perform or offer commercial services or products
of any kind using the Sequencing Kits is hereby granted. A license to use the technology
for commercial purposes is subject to a separate license agreement with GE Healthcare
Bio-Sciences AB. Please contact the Product Director, Mass Spectrometry and Sample
Handling, GE Healthcare Bio-Sciences AB, Björkgatan 30, SE-75184, Uppsala, Sweden for
details about how to obtain such a license.
2-D Electrophoresis
This version of ImageMaster has been developed by the Swiss Institute of Bioinformatics in
collaboration with GeneBio and GE Healthcare.
All third party trademarks are the property of their respective owners.
Principles and Methods
© 2010 General Electric Company—All rights reserved.
All goods and services are sold subject to the terms and conditions of sale of the company
within GE Healthcare which supplies them. A copy of these terms and conditions is available
on request. Contact your local GE Healthcare representative for the most current information.
For local office contact information,
please visit www.gelifesciences.com/contact
www.gelifesciences.com/protein-purification
GE Healthcare Bio-Sciences AB
Björkgatan 30
751 84 Uppsala
Sweden
GE Healthcare UK Limited Amersham Place
Little Chalfont
Buckinghamshire, HP7 9NA
UK
GE Healthcare Europe, GmbH
Munzinger Strasse 5
D-79111 Freiburg
Germany
GE Healthcare Bio-Sciences Corp.
800 Centennial Avenue, P.O. Box 1327
Piscataway, NJ 08855-1327
USA
GE Healthcare Bio-Sciences KK
Sanken Bldg., 3-25-1, Hyakunincho
Shinjuku-ku, Tokyo 169-0073
Japan
imagination at work
imagination at work
imagination at work
80-6429-60 AD 06/2010
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