Ettan DIGE System - McGill University

Ettan DIGE System - McGill University
GE Healthcare
Ettan DIGE System
User Manual
Ettan
Important user information
All users must read this entire user manual to fully
understand the safe use of Ettan DIGE System.
For safe use of Ettan DIGE System related products
described in this user manual, see corresponding
manuals.
WARNING!
The WARNING! sign highlights instructions that
must be followed to avoid personal injury. It is
important not to proceed until all stated
conditions are met and clearly understood.
CAUTION!
The Caution! sign highlights instructions that
must be followed to avoid damage to the
product or other equipment. It is important not
to proceed until all stated conditions are met
and clearly understood.
Note:
The Note sign is used to indicate information
important for trouble-free and optimal use of
the product.
Contents
1
Introduction
1.1
1.2
1.2.1
1.2.2
1.3
1.4
2
2.1.1
2.1.2
2.2
2.2.1
2.2.2
2.3
2.3.1
2.3.2
2.4
2.4.1
2.4.2
2.5
2.6
Ettan DIGE System User Manual .............................................................11
Ettan DIGE System related products manuals .................................12
Introduction .......................................................................................................13
2D analysis result variation ........................................................................... 13
Improvement of results by use of Ettan DIGE system ....................... 13
CyDye DIGE Fluor dyes ................................................................................14
CyDye DIGE Fluor minimal dyes.................................................................. 15
CyDye DIGE Fluor saturation dyes ............................................................. 17
The internal standard ...................................................................................18
Advantages of using an internal standard.............................................. 18
Examples of benefits using an internal standard................................. 19
Co-detection and matching using DeCyder 2D ...............................20
Intra-gel co-detection....................................................................................... 20
Inter-gel matching ............................................................................................. 21
Protein abundance ........................................................................................22
Statistical tests of protein abundance in DeCyder 2D ..................24
Experimental design
3.1
3.2
3.2.1
3.2.2
3.2.3
3.2.4
3.3
3.3.1
3.3.2
4
Analytical workflow........................................................................................... 10
Preparative workflow ....................................................................................... 11
DIGE concepts
2.1
3
Ettan DIGE System overview ....................................................................... 9
Workflows in 2D analysis .............................................................................. 9
Introduction .......................................................................................................25
Designing 2D DIGE experiments ..............................................................25
Internal standard sample on each gel ...................................................... 25
Biological replicates .......................................................................................... 25
Randomization of samples ............................................................................ 26
No gel replicates of the same sample is needed.................................. 26
Examples of experimental design ..........................................................26
CyDye DIGE Fluor minimal dyes.................................................................. 26
CyDye DIGE Fluor saturation dyes ............................................................. 27
Sample preparation and labeling
4.1
4.2
4.3
4.3.1
4.3.2
4.4
4.5
4.6
Introduction .......................................................................................................29
Workflow ............................................................................................................30
Sample preparation ......................................................................................30
Solution recommendations............................................................................ 30
Protocol................................................................................................................... 31
pH adjustment .................................................................................................32
Protein concentration determination ...................................................33
Internal standard preparation ..................................................................33
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Contents
4.7
4.7.1
4.7.2
4.7.3
4.7.4
4.8
4.9
5
5.1.1
5.2
5.2.1
5.3
5.3.1
5.3.2
5.4
5.5
7.5.1
7.6
7.6.1
7.7
General precautions for good results........................................................ 42
Workflow ............................................................................................................ 42
Sample application protocol selection...................................................... 42
Rehydration and sample application ................................................... 43
Rehydration of Immobiline DryStrips ........................................................ 43
Preparations for first dimension run including Cup loading ........... 45
First dimension isoelectric focusing (IEF) ............................................ 47
Recipes ............................................................................................................... 48
Ettan DALT electrophoresis system ...................................................... 51
Workflow ............................................................................................................ 52
Casting homogeneous gels ...................................................................... 52
Equilibration of focused Immobiline DryStrips ................................ 54
Loading of focused Immobiline DryStrips .......................................... 55
Second dimension SDS PAGE ................................................................... 56
Recipes ............................................................................................................... 58
Typhoon Variable Mode Imager ............................................................ 61
Workflow ............................................................................................................ 62
Cleaning Typhoon ......................................................................................... 62
Placing gels in Typhoon .............................................................................. 62
Scan parameters and scanning ............................................................. 64
Pre-scanning to identify a suitable PMT voltage .................................. 70
Cropping using ImageQuant TL .............................................................. 71
ImageQuant TL trouble shooting................................................................. 72
Ettan DIGE Imager ......................................................................................... 73
Image analysis
8.1
8.1.1
8.2
8.2.1
8.2.2
vi
Ettan IPGphor 3 Isoelectric Focusing System .................................. 41
Image acquisition
7.1
7.2
7.3
7.4
7.5
8
First dimension sample preparation .................................................... 38
Recipes ............................................................................................................... 38
Second dimension SDS PAGE
6.1
6.2
6.3
6.4
6.5
6.6
6.7
7
Preparation of CyDye DIGE Fluor dyes for labeling ............................ 33
Preparation of working dye solution ......................................................... 35
CyDye DIGE Fluor minimal dye labeling .................................................. 36
CyDye DIGE Fluor saturation dye labeling.............................................. 37
First dimension isoelectric focusing (IEF)
5.1
6
Labeling .............................................................................................................. 33
DeCyder 2D software .................................................................................. 75
Modules................................................................................................................... 75
ImageMaster 2D Platinum software ..................................................... 77
Structure................................................................................................................. 77
Image analysis workflow................................................................................ 77
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Contents
9
Preparative workflow
9.1
9.1.1
9.2
9.3
9.4
Introduction .......................................................................................................79
Staining of preparative gels........................................................................... 79
Sample preparation ......................................................................................81
First dimension isoelectric focusing (IEF) .............................................81
Second dimension SDS PAGE ....................................................................81
9.4.1
9.4.2
9.4.3
Clean and Bind-Silane treat the glass plates ......................................... 81
Attach reference markers ............................................................................... 83
Second dimension SDS PAGE........................................................................ 84
9.5
9.6
9.7
9.8
9.9
9.10
Post-staining with Deep Purple Total Protein Stain ........................84
Gel scanning .....................................................................................................85
Matching gels and creating a pick list ..................................................86
Spot picking the gel .......................................................................................86
Mass spectrometry analysis .....................................................................87
Recipes ................................................................................................................87
Appendix A Testing cell lysates for successful labeling
A.1
A.2
Testing new cell lysate for successful labeling................................... 91
Recipes................................................................................................................... 96
Appendix B Labeling of cell surface proteins
B.1
Selective labeling of cell surface proteins............................................. 97
Appendix C Reagents tested for compatibility with Ettan DIGE
system
C.1
List of reagents .................................................................................................. 99
Appendix D Trouble shooting guide
D.1
D.2
D.3
D.4
Sample preparation and labeling ........................................................... 103
First dimension electrophoresis............................................................... 105
Second dimension electrophoresis ........................................................ 106
Typhoon Variable Mode Imager results ............................................. 108
Index
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Contents
viii
Ettan DIGE System User Manual 18-1173-17 Edition AB
Introduction 1
1
Introduction
1.1
Ettan DIGE System overview
Ettan™ DIGE system is based on the technique of two-dimensional difference gel
electrophoresis (2D DIGE). It is a powerful tool for separating complex mixtures of
proteins by charge and size and for scanning and analyzing the resulting second
dimension SDS PAGE gel images for protein differences. Combining novel
proprietary technologies in fluorescence, sample multiplexing and image
analysis, Ettan DIGE System is a fully integrated system offering significant
benefits over classical second dimension SDS PAGE.
The system comprises CyDye™ DIGE Fluor minimal dyes, labeling kit for scarce
samples, Typhoon™ Variable Mode Imager, and DeCyder™ 2D software.
Alternatively, Ettan DIGE Imager™ can be used instead of Typhoon Variable Mode
Imager, and ImageMaster™ 2D Platinum software can be used instead of
DeCyder 2D software.
The use of CyDye DIGE Fluor minimal dyes enables multiplexing of up to three
separate protein mixtures on the same second dimension SDS PAGE gel. The
multiplexing capability of the 2D DIGE methodology enables the incorporation of
the same internal standard on every gel and thereby eliminates gel-to-gel
variation. Ensuring that each protein spot has its own internal standard is the only
way to remove gel-to-gel variation, thereby significantly increasing accuracy
and reproducibility.
To capitalize on this ability to multiplex, DeCyder 2D software and ImageMaster
2D Platinum software have been specifically designed for the Ettan DIGE system
to accurately measure very small protein differences with high confidence.
DeCyder 2D software and ImageMaster 2D Platinum software contains
proprietary algorithms that perform co-detection of differently labeled samples
within the same gel.
1.2
Workflows in 2D analysis
Two main workflows using Ettan DIGE System are presented:
•
Analytical workflow
•
Preparative workflow
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1 Introduction
1.2 Workflows in 2D analysis
1.2.1
Analytical workflow
The analytical workflow is used to find proteins of interest in an experiment. It
includes defining the experimental design, preparing the samples, performing 2D
separation of the proteins, scanning the gels and analyzing the gel images to find
proteins of interest. Fig 1-1 outlines an overview of the Ettan DIGE System
analytical workflow. See Chapters 4-8 for detailed information about the different
steps in the analytical workflow.
1. Set up the experimental design
Gel Cy2 Cy3 Cy5
1 ......... ......... .........
2
3
......... ......... .........
......... ......... .........
2. Prepare the samples
Unlabeled
Cy2
Cy3
Cy5
Labeling
Pool portion
Mix
Labeling
Internal standard
3. Perform first and second dimension electrophoresis
4. Scan the gels
Gel images
5. Perform image analysis (DeCyder 2D Software)
DIA
BVA
EDA
Proteins of interest
Fig 1-1. Analytical workflow in Ettan DIGE System. Labeling is performed using CyDye DIGE
Fluor minimal dyes. First dimension and second dimension SDS PAGE are performed using
Ettan IPGphor™ 3 and Ettan DALTtwelve or Ettan DALTsix. Scanning is performed using
Typhoon Variable Mode. Imager and image analysis is performed in DeCyder 2D software.
10
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Introduction 1
1.2.2
Preparative workflow
The preparative workflow is used to perform spot picking and protein
identification of the proteins of interest found in the analytical workflow. To
perform this, a pick list containing the coordinates for the proteins of interest to
pick is created and a pick gel (preparative gel) is prepared. See Chapter 9 for
detailed information about the different steps in the preparative workflow.
1.3
Ettan DIGE System User Manual
The scope of this user manual is to explain the central concept of the DIGE
technology, to give an understanding of the entire experimental workflow and an
overview of the Ettan DIGE related products available. Chapter 2 and 3 give a
background to the DIGE system and to experimental design, chapters 4 to 8 cover
the DIGE system analytical workflow, and chapter 9 contains special protocols for
DIGE system preparative workflow. The four appendices cover other important
information.
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1 Introduction
1.4 Ettan DIGE System related products manuals
1.4
Ettan DIGE System related products manuals
Because Ettan DIGE System comprises many different products, references to
the appropriate manuals for further details are made when necessary, see
Table 1-1 . The manuals are also available on the web (www.ettandige.com).
Table 1-1. Ettan DIGE System related products manuals.
12
Manual
Code no.
Ettan IPGphor 3 Control Software User Manual
11-0034-59
Ettan IPGphor 3 Instrument User Manual
11-0034-58
Ettan IPGphor Cup Loading Manifold User Manual
11-0034-60
2D Electrophoresis Principles and Methods
80-6429-60
Ettan DALTtwelve system User Manual
80-6476-53
Ettan DALTsix system User Manual
80-6492-49
Typhoon User´s Guide
63-0028-31
Ettan DIGE Imager User Manual
11-0036-59
ImageQuant TL User Manual
63-0050-82
DeCyder 2D Software User Manual
28-4010-06
DeCyder Extended Data Analysis Module User Manual
28-4010-07
ImageMaster 2D Platinum 6.0 User Manual
11-0034-38
Deep Purple Total Protein Stain Instructions
RPN6305PL
Ettan Spot Picker User Manual
18-1147-61
Ettan Digester User Manual
18-1167-31
Ettan Spotter User Manual
11-0003-41
Ettan Spot Handling Workstation User Manual
18-1153-55
Ettan DIGE System User Manual 18-1173-17 Edition AB
DIGE concepts 2
2
DIGE concepts
2.1
Introduction
2.1.1
2D analysis result variation
2D analysis experiments commonly address questions like protein level
differences caused by a disease state, drug treatment, life-cycle stage etc. Some
protein level differences studied are small and the results are affected by
experimental variation originating both from the system and from inherent
biological variation.
System related result variation
System related result variation may arise for two reasons:
1
Gel-to-gel variation, which can result from differences in electrophoretic
conditions between first dimension strips or second dimension gels, gel
distortions, sample application variation and user-to-user variation.
2
Variation due to user-specific editing and interpretation when using the data
analysis software.
Inherent biological variation
Inherent biological variation arises from intrinsic differences that occur within a
population. For example, differences from animal-to-animal, plant-to-plant or
culture-to-culture which have been subjected to identical conditions.
2.1.2
Improvement of results by use of Ettan DIGE system
There are three main factors enabling Ettan DIGE System to provide greater
accuracy than conventional 2D analysis:
1
Multiplexing, that is to run multiple samples on the same gel
2
Use of an internal standard for all proteins which can be run on all gels in a
set of experiments
3
Experimental designs unique to this technique, see chapter 3.
Compensation for system related result variation
System variation cannot be overcome when using conventional 1-color 2D
electrophoresis but by using Ettan DIGE System it is possible to minimize the gelto-gel variation effects on results.
•
In small experiments it is possible, by multiplexing, to run all samples on the
same gel and thereby completely eliminate gel-to-gel variation.
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2 DIGE concepts
2.2 CyDye DIGE Fluor dyes
•
In larger experiments a combination of multiplexing of samples and
inclusion of an internal standard within each gel all system related result
variation can be adjusted for. Any protein spot can be compared and
normalised to any other corresponding protein spot in the same or any
other gel, in which the same internal standard has been used.
Differentiation of inherent biological variation from induced biological change
Biological variation cannot be removed from any 2D electrophoresis
experiments. However, Ettan DIGE system allows the inherent biological variation
to be effectively differentiated from induced biological changes using highly
accurate measurement of protein abundance changes, an appropriate
experimental design and advanced statistical analysis.
The use of biological replicates in the experimental design ensures a true
measurement of induced biological differences above the background of
inherent biological variation. Ettan DIGE system is capable of detecting and
quantifying differences as small as 10% between samples (above system
variation) with greater than 95% statistical confidence.
2.2
CyDye DIGE Fluor dyes
Ettan DIGE system is based upon the specific properties of the CyDye DIGE Fluor
dyes. There are two different CyDye DIGE Fluor dyes available, CyDye DIGE Fluor
minimal dyes and CyDye DIGE Fluor saturation dyes. The saturation dyes are also
known as labeling kit for scarce samples. The key differences between CyDye
DIGE Fluor minimal and saturation dyes are summarized in Table 2-1 .
14
•
Use CyDye DIGE Fluor minimal dyes for normal applications. Multiplexing up
to three samples and labeling of 50 µg protein is possible.
•
Use CyDye DIGE Fluor saturation dyes when samples are precious or
available in very small amounts. Multiplexing up to two samples and
labeling of 5 µg protein is possible.
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DIGE concepts 2
Table 2-1. Summary of key differences between CyDye DIGE Fluor minimal and saturation
dyes.
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
Reconstituted stock at
2 mM (analytical gels) or
20 mM (preparative gels)
Reconstituted stock at 1 mM
No dilution of stock solution
Working concentration 0.4 mM
TCEP prior to labeling
No reduction step required
Reducing step
Protein labeling
Protein separation and
analysis
Labeling reaction at 37°C
Labeling reaction on ice
Labeling reaction quenched with
2x sample buffer
Labeling reaction quenched with
10 mM lysine
Labeling optimization by titrating TCEP
and dye, analysis on 1D (SDS PAGE) gel
Labeling optimization by comparing
labeled samples, analysis on 1D (SDS
PAGE) gel
Labeled proteins stable 1 month at
-70°C
Labeled proteins as stable as
unlabeled proteins at -70°C
No iodoacetamide equilibration step
prior to second dimension
electrophoresis
Iodoacetamide equilibration step
required prior to second dimension
electrophoresis
A Cy™3 labeled sample is used on
preparative gel for spot picking.
An unlabeled sample is used on
preparative gel for spot picking. Poststaining is required for matching to
analytical gels
2.2.1
CyDye DIGE Fluor minimal dyes
Chemical description
CyDye DIGE Fluor minimal dyes are three spectrally resolvable dyes (Cy™2, Cy3
and Cy5) matched for mass and charge. Each CyDye DIGE Fluor minimal dye,
when coupled to a protein, will add 450 Da to the mass of the protein. This mass
shift does not effect the pattern visible on a second dimension SDS PAGE gel. A
protein labeled with any of the CyDye DIGE Fluor minimal dyes will migrate to the
same position on the second dimension SDS PAGE gel, thus making multiplexing
possible.
Sensitivity
The dyes afford great sensitivity down to 25 pg of a single protein, and a linear
response to protein concentration up to five orders of magnitude (105). In
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2 DIGE concepts
2.2 CyDye DIGE Fluor dyes
comparison, silver stain detects 1–60 ng of protein with a dynamic range of less
than two orders of magnitude.
Protein labeling
CyDye DIGE Fluor minimal dyes have an NHS ester reactive group, and are
designed to form a covalent bond with the epsilon amino group of lysine residues
in proteins via an amide linkage. The dye is added to the protein such that the
amount of dye is limiting within the labeling reaction. The lysine amino acid in
proteins carries an intrinsic +1 charge at neutral or acidic pH. CyDye DIGE Fluor
minimal dyes also carry a +1 charge which, when coupled to the lysine, replaces
the lysine’s +1 charge with its own, ensuring that the pI of the protein does not
significantly alter.
With CyDye DIGE Fluor minimal dyes 50 µg protein is labeled in each reaction. The
ratio used ensures that the dyes label approximately 1–2% of lysine residues so
each labeled protein carries only one dye label and is visualised as a single
protein spot. The CyDye DIGE Fluor minimal dyes therefore only label a small
proportion of the total protein in a sample. For that reason, this type of labeling
has been called minimal labeling.
Dye +
Dye +
Fig 2-1. Schematic of the minimal labeling reaction. CyDye DIGE Fluor minimal dye containing NHS ester active group covalently binds to the lysine residue of a protein via an amide
linkage.
16
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DIGE concepts 2
2.2.2
CyDye DIGE Fluor saturation dyes
Chemical description
CyDye DIGE Fluor saturation dyes (also known as labeling kit for scarce samples)
are two spectrally resolvable dyes (Cy3 and Cy5) matched for mass and charge.
Each CyDye DIGE Fluor saturation dye, when coupled to a protein, will add
approximately 677 Da to the mass of the protein. A protein labeled with any of
the CyDye DIGE Fluor saturation dyes will migrate to the same position on the
second dimension SDS PAGE gel, in this way making multiplexing possible.
Sensitivity
The dyes afford great sensitivity with detection lower than 25 pg of a single
protein, and a linear response to protein concentration up to five orders of
magnitude (105).
Protein labeling
CyDye DIGE Fluor saturation dyes have an maleimide reactive group which is
designed to form a covalent bond with the thiol group of cysteine residues in
proteins via a thioether linkage. CyDye DIGE Fluor saturation dyes have a neutral
charge and will not affect the pI of the labeled protein.
Reduction step
TCEP, 1 h 37 °C
S
S
protein
HS-
protein
pH 8.0
Coupling step
Dye
HS-
O
O
N
H
N
protein
Dye
37 °C, 30 min
pH 8.0
O
O
O
N
H
N
O
S
protein
Maleimide reactive group
Fig 2-2. Schematic of the saturation labeling reaction.
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2 DIGE concepts
2.3 The internal standard
With CyDye DIGE Fluor saturation dyes 5 µg protein is labeled in each reaction.
The dye is added to the protein under such conditions that all available cysteine
residues of the proteins are labeled. For that reason, this type of labeling has been
called saturation labeling. To achieve optimum labeling a high dye-to-protein
ratio is required.
2.3
The internal standard
The multiplexing capability of the 2D DIGE methodology enables the
incorporation of the same internal standard on every second dimension SDS
PAGE gel. The internal standard is a pool of all the samples within the experiment,
and therefore contains every protein from every sample.
The internal standard is used to match and normalize the protein patterns across
different gels thereby negating the problem of inter-gel variation, a common
problem in standard “one sample per gel” 2D electrophoresis experiments.
The internal standard allows accurate quantitation of differences between
samples, with an associated statistical significance. Quantitative comparisons of
protein between samples are made on the relative change of each protein spot
to its own in-gel internal standard. It enables accurate, statistical quantification
of induced biological change between samples. The 2D DIGE methodology is the
only technique to enable accurate standardized quantitation.
2.3.1
Advantages of using an internal standard
The recommended protocol for experiments with more than three samples
includes an internal standard that is run on all gels within an experiment together
with up to two different labeled protein samples.
Linking every sample in all gels to a common internal standard offers a number
of advantages:
18
•
Accurate quantification and accurate spot statistics between gels
•
Increased confidence in matching between gels
•
Flexibility of statistical analysis depending on the relationship between
samples
•
Separation of induced biological change from system variation and
inherent biological variation
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DIGE concepts 2
2.3.2
Examples of benefits using an internal standard
The examples below illustrate the benefits of an internal standard. Fig 2-3 shows
the theoretical scan results of two gels. Each gel contained two protein samples
labeled with CyDye DIGE Fluor Cy3 or Cy5 minimal dyes and the same pooled
internal standard sample labeled with CyDye DIGE Fluor Cy2 minimal dye.
If the gels illustrated in Fig 2-3 were analyzed without an internal standard, the
conclusion would be that the volume of the highlighted protein spot in samples 1
and 2 has remained the same but is increased slightly in sample 3 and further in
sample 4. However, reference to the internal standard shows that gel-to-gel
variation has resulted in an increased spot volume in gel B compared to gel A. This
means that instead of an increasing trend in spot volume from samples 1 to 4, the
relative volume of the protein spot in sample 3 is reduced in comparison to
samples 1, 2 and 4 where the spot volume ratios are identical.
Gel
Cy2 (Standard)
Cy3
Cy5
A
Pool samples 1–4
Sample 1 - untreated
Sample 2 - treated
B
Pool samples 1–4
Sample 3 - treated
Sample 4 - untreated
Fig 2-3. Example to illustrate the benefits of an internal standard in correctly identifying
differences between samples 1, 2, 3 and 4. The right panel shows the different results
achieved of the volumes of the protein spots without and with an internal standard.
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2 DIGE concepts
2.4 Co-detection and matching using DeCyder 2D
2.4
Co-detection and matching using DeCyder 2D
To capitalize on the ability to multiplex and use an internal standard, DeCyder 2D
software has been specifically designed for the Ettan DIGE system. DeCyder 2D
contains proprietary algorithms that perform co-detection of differently labeled
samples within the same gel. DeCyder 2D also permits automated detection,
background subtraction, quantitation, normalization, internal standardization
and inter-gel matching. The benefits are low user interaction, high throughput
and low experimental variation. For an introduction of DeCyder 2D, see Chapter
8. For a detailed guide, see DeCyder 2D Software User Manual.
To compare protein spot volumes across a range of experimental samples and
gels, two distinct steps are required:
•
Intra-gel co-detection of sample and internal standard protein spots
•
Inter-gel matching of internal standard samples across all gels within the
experiment
Both of these analysis steps can be performed with minimal user intervention by
DeCyder 2D.
2.4.1
Intra-gel co-detection
Three scans will be made of each gel, Cy2, Cy3 and Cy5 scans. Scanned images
of each sample and the internal standard are overlaid in DeCyder 2D. The
algorithms within the software co-detect the spots present in each scan,
effectively identifying the position of each spot within the gel (Fig 2-4). The spot
boundaries that result are identical for each image in the gel. This minimizes
variation from detection and background subtraction, with the added benefit that
every protein in the sample is intrinsically linked to the corresponding protein
spot in the internal standard sample.
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DIGE concepts 2
Fig 2-4. Intra-gel co-detection - All samples are co-detected with the internal standard.
2.4.2
Inter-gel matching
Experimental design ensures that each gel contains the same internal standard.
This enables inter-gel comparisons of spot abundance. Before this can be done,
it is important to ensure that the same protein spots are compared between gels.
DeCyder 2D achieves this using the internal standard to match the position of
each protein across all gels within the experiment. The internal standard image
with the most detected spots is assigned as the 'Master'. Following co-detection,
each image has a spot map species. The spot map species for the internal
standard assigned as the Master, is used as a template to which all remaining
spot map species for the other internal standards (intrinsically linked to their codetected sample images) are matched (Fig 2-5).
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2 DIGE concepts
2.5 Protein abundance
Fig 2-5. Inter-gel matching - only the internal standards need to be matched. These are
derived from the same sample which aids matching.
2.5
Protein abundance
Once the protein spots have been matched, the ratio of protein abundance
between samples can be determined.
The use of an identical internal standard within all the experimental gels enables
a comparison of protein abundance between samples on different gels. This is
performed by comparison of the ratios of sample:standard, rather than direct
comparison of raw spot volumes.
In this way differences in spot intensity that may arise due to experimental
factors during the process of 2D electrophoresis, such as protein loss during
sample transfer, will be the same for each sample within a single gel, including the
internal standard. This means that the relative ratio of sample:standard will not
be affected by such variation due to experimental factors.
Spot volume (i.e. the sum of the pixel values within a spot minus background) for
each experimental sample is compared directly to the internal standard by
DeCyder 2D. Spot ratios are calculated (volume of secondary image spot / volume
of primary image spot) indicating the change in spot volume between the two
images. The protein abundance for each spot in each sample is then expressed
as a (normalized) ratio relative to the internal standard (the primary image) e.g.
[Cy3 sample 1:Cy2 standard] and [Cy5 sample 2:Cy2 standard]. From this
analysis, cross-sample comparisons can be made, see Table 2-2 .
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DIGE concepts 2
Table 2-2. Ratio of (sample):(internal standard) for a single protein of interest.
Sample
Ratio of (sample):(internal standard)
for a single protein of interest
A1
-2.1
A2
-2.4
A3
-1.9
A4
-2.5
B1
2.6
B2
2.5
B3
2.2
B4
2.4
Note: Down regulation of protein abundance relative to the internal standard is
denoted by a negative prefix, for example, a two-fold decrease, or a
conventional ratio of 0.5 is displayed as -2.0.
DeCyder 2D software can graphically display the relative abundance of each
protein against the normalized internal standard, see Fig 2-6.
Fig 2-6. Plot of sample ratios relative to normalized internal standards.
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23
2 DIGE concepts
2.6 Statistical tests of protein abundance in DeCyder 2D
It is possible to compare the protein abundance for a protein of interest in
different samples. The ratio of (sample A):(sample B) for the protein of interest
shown in Table 2-2 and Fig 2-6 can be calculated, see Table 2-3 . The result shows
that the protein of interest is down-regulated approximately five-fold in sample A
compared to sample B.
Table 2-3. Ratio of (sample A):(sample B) calculated from (sample):(standard) ratios shown
in Fig 2-6.
Sample
2.6
Ratio of (sample A):(sample B)
A1:B1
-5.4
A2:B2
-6.0
A3:B3
-4.2
A4:B4
-6.0
Statistical tests of protein abundance in DeCyder 2D
Statistical tests are important and give the user a level of confidence by taking
into account the inherent biological variation within a group compared to the
induced difference between groups and assigning a confidence rating as to
whether this change is above the biological variation.
Statistical tests can then be applied to the data in DeCyder 2D, for example,
Student’s T-test and ANOVA. The statistical tests compare the average ratio and
variation within each group to the average ratio and variation in the other groups
to see if any change between the groups is significant. If using the Extended Data
Analysis (EDA) module of DeCyder 2D, additional multivariate statistical analyses
such as Principal Component Analysis (PCA), Pattern Analysis and Discriminant
Analysis can be performed.
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Experimental design 3
3
Experimental design
3.1
Introduction
Prior to commencing practical work, experimental design needs to be carefully
considered. This chapter addresses experimental design unique to 2D DIGE
analysis for optimal data analysis using DeCyder 2D software.
3.2
Designing 2D DIGE experiments
When designing 2D DIGE experiments, the following recommendations should be
considered:
1
Inclusion of an internal standard sample on each gel
2
The requirement for biological replicates such as multiple cultures, tissue etc.
3
Randomization of samples to produce unbiased results, thus conforming
with best experimental practice
4
No gel replicates of the same sample is needed
3.2.1
Internal standard sample on each gel
It is recommended that an internal standard is run on all gels within an
experiment as it is then possible to minimize effects of system related result
variation, see section 2.3. The analysis of results then allows the inherent
biological variation to be effectively differentiated from induced biological
changes using appropriate experimental design and statistical analysis.
Quantitative comparisons of protein between samples are made on the relative
change of each protein spot to its own in-gel internal standard. This removes gelto-gel (system) variation, a common problem with conventional one sample per
gel 2D studies. It also enables accurate, statistical quantification of induced
biological change between samples. Ettan DIGE System is the only protein
difference analysis technique that utilises this approach.
3.2.2
Biological replicates
It is strongly advised that biological replicates are included in every group. By
increasing the number of biological replicates it is possible to get an accurate
measurement of the change due to a treatment or disease that is significant
above a baseline of inherent biological variation. Gel replicates of the same
biological sample will not deliver this information. Without biological replicates,
results may not be biologically relevant and it is often only possible to conclude
that differences in results are above system variation.
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3 Experimental design
3.3 Examples of experimental design
3.2.3
Randomization of samples
Randomization of samples across gels removes any bias from the experiments
such as experimental conditions, sample handling and labeling, ensuring that
results from 2D DIGE experiments are accepted by peers. Even if the system
related result variation is low using Ettan DIGE System it is good laboratory
practice to distribute individual experimental samples evenly between different
CyDye DIGE Fluor dyes and different gels to avoid for example systematic errors.
See Section 3.3 for examples.
3.2.4
No gel replicates of the same sample is needed
As the result variation using Ettan DIGE System is so low due to the internal
standard and method of analysis, any system variation will by far be outweighed
by the inherent biological variation. However, gel replicates can be included if
desired.
3.3
Examples of experimental design
In order to maximize the value of CyDye DIGE Fluor dyes and DeCyder 2D
software, it is important to carefully consider the experimental testing regime.
Two examples using CyDye DIGE Fluor minimal dyes and CyDye DIGE Fluor
saturation dyes (sections 3.3.1 and 3.3.2, respectively), are presented below to
illustrate examples of experimental design.
3.3.1
CyDye DIGE Fluor minimal dyes
Comparison of protein abundance between three differently treated samples
(A-C) each with four biological replicates using:
•
CyDye DIGE Fluor Cy2, Cy3, and Cy 5 minimal dyes
•
Internal standard labeled with CyDye DIGE Fluor Cy2 minimal dye
•
A design with randomized sample labeling of either CyDye DIGE Fluor Cy3
or Cy5 minimal dyes is strongly recommended. Samples to be evenly
distributed between the CyDye DIGE Fluors Cy3 and Cy5 and between gels
Experimental set up
• Mix 50 µg of each of the 12 samples (A1-A4, B1-B4 and C1-C4) together to
create 600 µg of the internal standard, and label with CyDye DIGE Fluor Cy2
minimal dye
26
•
Individually label 50µg of samples A1 - A4, B1 - B4 and C1 - C4 with either
CyDye DIGE Fluor Cy3 or Cy5 minimal dye in a randomized design
•
Six gels are required, loaded as described in Table 3-1
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Experimental design 3
Table 3-1. Gels to be run for Case study 1: Samples labeled with CyDye DIGE Fluor minimal
dyes.
Gel
Cy2 (Internal standard)
Cy3
Cy5
1
50 µg (4.17µg each of
samples A1-4, B1-4, C1-4)
50 µg sample B2
50 µg sample C1
2
50 µg (4.17µg each of
samples A1-4, B1-4, C1-4)
50 µg sample A1
50 µg sample B3
3
50 µg (4.17µg each of
samples A1-4, B1-4, C1-4)
50 µg sample C3
50 µg sample A4
4
50 µg (4.17µg each of
samples A1-4, B1-4, C1-4)
50 µg sample A3
50 µg sample C2
5
50 µg (4.17µg each of
samples A1-4, B1-4, C1-4)
50 µg sample B4
50 µg sample A2
6
50 µg (4.17µg each of
samples A1-4, B1-4, C1-4)
50 µg sample C4
50 µg sample B1
Total gels = 6
Note: By using three dyes instead of two the number of gels required is halved.
The amount of material required is also reduced as half the amount of
internal standard is used (six gels instead of twelve gels).
3.3.2
CyDye DIGE Fluor saturation dyes
Comparison of protein abundance between three differently treated samples (AC) each with four biological replicates using:
•
CyDye DIGE Fluor Cy3 and Cy5 saturation dyes
•
Internal standard labeled with CyDye DIGE Fluor Cy3 saturation dye
•
All experimental samples labeled with the same dye (CyDye DIGE Fluor Cy5
saturation dye)
•
Twelve gels are required, loaded as described in Table 3-1
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3 Experimental design
3.3 Examples of experimental design
Experimental set up
• Mix 5 µg of each of the 12 samples (A1-A4, B1-B4 and C1-C4) together to
create 60 µg of the internal standard
•
Label the internal standard with CyDye DIGE Fluor Cy3 saturation dye
•
Individually label 5 µg of samples A1 - A4, B1 - B4 and C1 - C4 with CyDye
DIGE Fluor Cy5 saturation dye
•
Twelve gels are required, loaded as described in Table 3-2
Table 3-2. Gels to be run for Case study 2: Samples labeled with CyDye DIGE Fluor
saturation dyes.
Gel
Cy3 Standard
Cy5
1
5 µg (4.17µg each of A1-4, B1-4, C1-4)
5 µg sample B4
2
5 µg (4.17µg each of A1-4, B1-4, C1-4)
5 µg sample C2
3
5 µg (4.17µg each of A1-4, B1-4, C1-4)
5 µg sample A4
4
5 µg (4.17µg each of A1-4, B1-4, C1-4)
5 µg sample A2
5
5 µg (4.17µg each of A1-4, B1-4, C1-4)
5 µg sample C4
6
5 µg (4.17µg each of A1-4, B1-4, C1-4)
5 µg sample B2
7
5 µg (4.17µg each of A1-4, B1-4, C1-4)
5 µg sample A1
8
5 µg (4.17µg each of A1-4, B1-4, C1-4)
5 µg sample C3
9
5 µg (4.17µg each of A1-4, B1-4, C1-4)
5 µg sample B1
10
5 µg (4.17µg each of A1-4, B1-4, C1-4)
5 µg sample A3
11
5 µg (4.17µg each of A1-4, B1-4, C1-4)
5 µg sample C1
12
5 µg (4.17µg each of A1-4, B1-4, C1-4)
5 µg sample B3
Total gels = 12
Note: It is possible to use CyDye DIGE Fluor minimal dyes in the same manner
labeling only with two dyes.
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Sample preparation and labeling 4
4
Sample preparation and labeling
4.1
Introduction
The preparation of a cell lysate and subsequent labeling with CyDye DIGE Fluor
minimal dyes or CyDye DIGE Fluor saturation dyes is detailed in this chapter. The
reagents and conditions stated here are those which have been found to be the
most consistently useful across many sample types. However, there will be cases
where some individual optimization of lysis conditions is required. See Section 2.2
for information of CyDye DIGE Fluor minimal dyes and CyDye DIGE Fluor
saturation dyes and Chapter 9 for special changes in protocols when running
preparative second dimension SDS PAGE for spot picking of proteins of interest.
Sample preparation for analytical gels
Unlabeled
Cy2
Cy3
Cy5
Cells
Protein extraction
Labeling with CyDye DIGE Fluor
minimal dyes
Samples
Pool portion
Mix
Labeling with CyDye DIGE Fluor
minimal dyes
Internal standard
Fig 4-1. Sample preparation and labeling with CyDye DIGE Fluor minimal dyes.
Recommended protocols for preparation of a cell lysate and labeling are
presented in sections 4.3 to 4.8 and recipes for recommended buffers and
solutions are given in section 4.9.
Note: Some standard methods for preparation of protein samples for
conventional 2D electrophoresis may not be compatible with Ettan DIGE
system.
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4 Sample preparation and labeling
4.2 Workflow
4.2
Workflow
Workflow for sample preparation and labeling:
1
Sample preparation: Prepare cell lysates compatible with CyDye DIGE Fluor
minimal dye or CyDye DIGE Fluor saturation dye labeling.
2
Adjust the pH of the cell lysates.
3
Determine cell lysates protein concentration.
4
Prepare an internal standard.
5
Label protein samples using CyDye DIGE Fluor minimal dyes or CyDye DIGE
Fluor saturation dyes.
6
Prepare labeled protein samples for first dimension isoelectric focusing.
4.3
Sample preparation
The sample preparation protocol in this section is designed to use cell cultures as
starting material.
For sample preparation protocols for other starting material, see
www.ettandige.com.
4.3.1
Solution recommendations
Cell wash solution
It is recommended to use the cell wash solution. As an alternative 0.5 X phosphate
buffered saline (PBS) can be used. However, if PBS is used it must be completely
removed since it may cause issues with high salt load during electrophoresis.
Any other wash solutions should be tested for compatibility with the labeling step
in controlled experiments. The cell wash solution used should not lyse the cells,
but it should dilute and remove any growth media, or reagents that might affect
CyDye DIGE Fluor dye labeling process.
Cell lysis solution
It is recommended to use the cell lysis solution. Alternatively, buffers such as Tris
or Bicarbonate can be used in the protein solution. The solution should be at a
concentration of approximately 30 mM. Higher concentration may affect
isoelectric focusing.
Note: Ensure that the pH remains between pH 8.0–9.0 in the cell lysate, by adding
the cell lysis solution. Failure to include a suitable buffer will mean that the
pH of the solution may fall below pH 8.0 resulting in little or no protein
labeling.
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Sample preparation and labeling 4
Note: The cell lysis solution should not contain any primary amines, such as
ampholytes or thiols (e.g. DTT) as these will compete with the proteins for
CyDye DIGE Fluor dyes resulting in fewer dye labeled proteins. This
decrease in labeling efficiency may affect the data after scanning and spot
detection.
4.3.2
Protocol
The example of protocol presented is adapted to Escherichia coli cell culture.
Other wash solution might be more appropriate for different cell types.
Approximately 4×1010 E. coli cells will result in 5-10 mg of protein in a total volume
of 1 ml of standard cell lysis solution.
1
Pellet the cells in a suitable centrifuge at +4°C.
2
Pour off all growth media, taking care not to disturb the cell pellet.
3
Re-suspend the cell pellet in 1 ml of standard cell wash solution in a
microfuge tube.
4
Pellet the cells in a bench-top microfuge at 12 000 × g for 4 min at +4ºC.
Remove and discard the supernatant.
5
Repeat steps 3 and 4 at least three times. Ensure all the cell wash solution
has been removed.
6
Re-suspend the washed cell pellet in 1 ml of standard cell lysis solution
and leave on ice for 10 min.
Note: If the protein concentration is less than 5 mg/ml after protein
quantitation, see section 4.5, re-suspend cells in a smaller volume
of lysis solution in subsequent experiments. Alternatively,
precipitate proteins using Ettan 2D Clean-Up Kit (code no. 80-648451), and re-suspend in a smaller volume of cell lysis solution.
7
Keep the cells on ice and sonicate intermittently until the cells are lysed.
8
Centrifuge the cell lysate at +4°C for 10 min at 12 000 × g in a
microcentrifuge.
9
Transfer supernatant to a labeled tube. This is the cell lysate. Discard the
pellet.
10 Remove contaminating substances with Ettan 2D Clean-Up Kit. This
procedure improves the labeling efficiency by removal of endogenous
small molecules. It also may improve spot resolution and increase the
number of spots detected.
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4 Sample preparation and labeling
4.4 pH adjustment
11 Check the pH of the cell lysate and adjust if necessary, see section 4.4.
The cell lysate can now be stored in aliquots, at –70 oC until protein yield
is to be determined.
Note: When using Ettan 2D Clean-Up Kit the pH drops considerably and
adjustment of pH to 8-9 is required.
4.4
pH adjustment
1
Check the pH of the cell lysate (protein sample) by spotting a small
volume (1-3 µl) on a pH indicator strip.
2
Optimal cell lysate pH is 8.5 for CyDye DIGE Fluor minimal dyes and 8.0
for CyDye DIGE Fluor saturation dyes.
Note: The use of cell lysates with optimal pH as described above is very
important. A lower pH than optimal will make labeling ineffective
and a higher pH than optimal will make the labeling unspecific.
3
Adjust the pH if the pH of the cell lysate is outside the desired range.
Normally, the pH needs to be increased.
4
To increase the pH in the cell lysate, make some more cell lysis solution
with tris base (without the protein) with pH 9.5 or higher.
5
Add aliquots of the new lysis solution to the cell lysate. This will gradually
increase the pH of the cell lysate. Stop when the pH of the protein sample
is at pH 8.5 (for minimal labeling).
Alternatively, the pH of the lysate can be increased to pH 8.5 by careful
addition of dilute sodium hydroxide (50 mM or higher).
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Sample preparation and labeling 4
4.5
Protein concentration determination
The concentration of protein in all lysates should be determined using a suitable
protein assay, compatible with detergents and thiourea if these are present in the
cell lysate.
Protein Determination Reagent (USB, code no. 30098) or Ettan 2D Quant Kit (code
no. 80-6483-56) are both suitable for this activity.
4.6
Internal standard preparation
The internal standard is created by pooling an aliquot of all biological samples in
the experiment and labeling it with one of the CyDye DIGE Fluor dyes (usually Cy2
when using CyDye DIGE Fluor minimal dyes and Cy3 when using CyDye DIGE
Fluor saturation dyes).
The internal standard is then run on every single gel along with each individual
sample. This means that every protein from all samples will be represented in the
internal standard, which is present on all gels. In this way every protein spot on
all gels will have an internal standard.
Note: Sufficient internal standard must be prepared to allow enough to be
included on every gel in the experiment.
4.7
Labeling
The protocols for labeling cell lysate samples with CyDye DIGE Fluor minimal and
saturation dyes are different. However, the preparation of dyes, including
reconstituting, and making working dye solutions are similar. Follow protocols in
sections 4.7.1 and 4.7.2, for both types of dyes but use the appropriate section of
4.7.3 and 4.7.4.
Note: It is recommended that all new cell lysates or samples containing chemical
components that has not been approved for DIGE use are checked for
successful labeling, see Appendix A.
4.7.1
Preparation of CyDye DIGE Fluor dyes for labeling
The reconstitution and storage of CyDye DIGE Fluor minimal and saturation dyes
is important to the success of sample labeling. If reagents, such as
dimethylformamide (DMF) are of a low quality, or the CyDye DIGE Fluor minimal
dyes are incorrectly stored, protein labeling will not be efficient.
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4 Sample preparation and labeling
4.7 Labeling
Reconstituting CyDye DIGE Fluor dye in dimethylformamide
CyDye DIGE Fluor minimal and saturation dyes are supplied as a solid powder
and are reconstituted in dimethylformamide (DMF) giving a concentration of 1
nmol/µl. After reconstitution in DMF the dye will give a deep color; Cy2-yellow,
Cy3-red, and Cy5-blue.
Note: It is recommended that a new bottle of DMF is opened every 3 months. The
DMF must be high quality anhydrous (Specification: <0.005% H2O, ≥ 99.8%
pure, Sigma Aldrich 22,705-6), and every effort should be taken to ensure it
is not contaminated with water.
Note: DMF, once opened, will start to degrade generating amine compounds,
which will react with the CyDye DIGE Fluor minimal dyes reducing the
concentration of dye available for protein labeling.
1
Take the CyDye DIGE Fluor minimal dye from the –20 ºC freezer, spin
briefly to ensure that the powder is at the bottom of the tube, and leave
to warm for 5 min at room temperature without opening. This will prevent
exposure of the dye to condensation which may cause hydrolysis.
2
Take a small volume of DMF from its original container and dispense into
a microfuge tube.
3
From this tube remove the specified volume of the aliquoted DMF (see
specification sheet supplied with the CyDye DIGE Fluor dye) and add to
each new vial of dye. Recommendations for:
•
CyDye DIGE Fluor minimal dyes: 25 µl DMF to 25 nmol of dye (1 mM)
•
CyDye DIGE Fluor saturation dyes: 50 µl DMF to 100 nmol of dye (2
mM)
4
Replace the cap on the microfuge tube containing the dye and vortex
vigorously for 30 seconds to dissolve the dye.
5
Centrifuge the microfuge tube for 30 seconds at 12 000 × g in a benchtop
microfuge.
CyDye DIGE Fluor saturation dye stock solution (2 mM) can be used with no
further dilutions. Once reconstituted, the saturation dye stock solution is stable
for three months.
CyDye DIGE Fluor minimal dye stock solution (1 nmol/µl) is prepared but should
be diluted before use. Once reconstituted, the minimal dye stock solution is stable
for three months or until the expiry date on the container, whichever is sooner.
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Sample preparation and labeling 4
Note: Check that the dye solution has an intense color. During transport, the dye
powder may spread around the inside surface of the tube (including the lid).
If the dye has not an intense color, then pipette the solution around the tube
(and lid) to ensure resuspension of dye. Vortex and spin down.
For dilution of the CyDye DIGE Fluor minimal dye stock solution:
1
Dilute the minimal dye stock solution to working dye solution
concentration, see section 4.7.2.
2
The dye stock solution should be stored in a light excluding container at
–20ºC. Return to freezer as soon as possible after use. The dye stock
solution is stable for up to three months at –20ºC.
4.7.2
Preparation of working dye solution
Amount of dye required for labeling
It is recommended that 400 pmol of dye is used to label 50 µg of protein. If
labeling more than 50 µg of protein then the dye:protein ratio must be
maintained for all samples in the same experiment. Other dye:protein ratios can
be used but must be optimized for the sample by testing the labeling on an
SDS PAGE gel, see Appendix A.
Dilution of dye stock solution
Prior to labeling, the dye stock solution is diluted with DMF (see above) to a
working dye solution. A concentration of 400 pmol/µl is recommended.
Note: The working dye solution is only stable for one week at –20ºC.
1
Briefly spin down the dye stock solution in a microcentrifuge.
2
Add 2 µl dye stock solution to 3 µl DMF.
3
Ensure all dye is removed from the pipette tip by pipetting up and down
the working dye solution several times.
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4 Sample preparation and labeling
4.7 Labeling
4.7.3
CyDye DIGE Fluor minimal dye labeling
The dye labeling reaction is designed to be simple and should take about 45 min
to perform.
Recommended conditions:
•
It is very important that the cell lysate (protein sample) has pH 8.5 in order to
achieve an efficient labeling, see section 4.4.
•
Dye to protein ratio of 400 pmol dye:50 µg protein. If the ratio of dye:protein
is too low, sensitivity may be decreased and if the ratio of dye:protein is too
high, there is a possibility of multiple dye molecules per protein and this
could lead to multiple spots per protein on the gel.
•
Protein concentration of 5-10 mg/ml in the cell lysate. However, samples
containing 1 mg/ml protein have been successfully labeled using the
protocol below.
Protocol
The protocol illustrates labeling of a cell lysate using 400 pmol of dye to label
50 µg of protein.
1
Add a volume of protein sample equivalent to 50 µg to a microfuge tube.
Bulk labeling reactions can be performed by scaling up as required.
2
Add 1 µl of working dye solution (400 pmol) to the microfuge tube
containing the protein sample.
3
Mix dye and protein sample thoroughly by pipetting and vortexing.
4
Centrifuge briefly in a microcentrifuge to collect the solution at the
bottom of the tube. Leave on ice for 30 min in the dark.
5
Add 1 µl of 10 mM lysine to stop the reaction. Mix and spin briefly in a
microcentrifuge. Leave for 10 min on ice, in the dark.
Labeling is now finished. The labeled samples can be processed immediately or
stored for up to 3 months at -70°C in the dark.
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Sample preparation and labeling 4
4.7.4
CyDye DIGE Fluor saturation dye labeling
The dye labeling reaction is designed to be simple and should take about 2 h to
perform.
Recommended conditions:
•
It is very important that the cell lysate (protein sample) has pH 8.0 in order to
achieve an efficient labeling, see section 4.4.
•
TCEP:dye concentration ratio should be kept at a 1:2 ratio to ensure efficient
labeling.
•
Protein concentration of 0.55-10 mg/ml in the cell lysate.
•
For samples containing proteins of interest with high cysteine content, more
TCEP (reduces disulphide bonds) and more dye (for labeling thiol groups) are
required.
Protocol
1
Add a volume of protein sample equivalent to 5 µg to a microfuge tube.
2
Make up to 9 µl with cell lysis solution.
3
Add 1 µl 2 mM TCEP.
4
Mix vigorously by pipetting and spin.
Note: Since cell lysates are viscous it is important to mix samples
thoroughly in this and all following mixing steps to avoid nonuniform labeling.
5
Incubate at 37°C for 1h in the dark.
6
Add 2 µl 2 mM CyDye DIGE Fluor saturation dye solution.
Note: Label the pooled protein internal standard sample with Cy3 and
the experimental protein sample with Cy5.
7
Mix vigorously by pipetting and spin.
8
Incubate at 37°C for 30 min in the dark.
9
Stop the reaction by adding an equal volume of 2x sample buffer.
10 Mix vigorously by pipetting and spin.
Labeling is now finished. The labeled samples can be processed immediately or
stored for 1 month at -70°C in the dark.
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4 Sample preparation and labeling
4.8 First dimension sample preparation
4.8
First dimension sample preparation
The main difference between conventional 2D electrophoresis and Ettan DIGE
system is that the latter will enable up to three different protein samples to be run
on a single 2D gel. To achieve this you need to mix the differently labeled protein
samples BEFORE the first dimension run.
Protocol
It is recommended that 50 µg (for CyDye DIGE Fluor minimal dyes) or 5 µg (for
CyDye DIGE Fluor saturation dyes) of each labeled protein sample is combined for
each gel.
1
Combine the two (for CyDye DIGE Fluor saturation dyes) or three (for
CyDye DIGE Fluor minimal dyes) labeled samples into a single microfuge
tube and mix. One of these samples should be the pooled internal
standard.
2
For CyDye DIGE Fluor minimal dye labeled samples: Add an equal
volume of 2× sample buffer to the labeled protein samples and leave on
ice for 15 minutes.
The samples are now ready for the first dimension isoelectric focusing step.
Note: After adding 2× sample buffer and incubating on ice it is recommended that
the sample is run immediately on Immobiline™ DryStrips. Proceed to
Chapter 5, First dimension isoelectric focusing (IEF).
4.9
Recipes
Cell wash solution
Reagent
Quantity
Final concentration
Tris (100 mM, pH 8.0)
5.0 ml
10 mM
Magnesium acetate (1 M)
0.25 ml
5 mM
Make up to 50 ml with distilled water
Store at 4 ºC. Stable for 1 month.
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Sample preparation and labeling 4
Cell lysis solution
1 Decide whether to use cell lysis solution 1 or 2. The cell lysis solution option
1 is very similar to option 2 except that thiourea is added, which has been
shown to solubilize many more proteins, especially membrane proteins.
2
Adjust the whole solution to pH 8.5 using dilute HCl. Confirm the pH of your
cell lysis solution by spotting 5 µl on a pH indicator strip.
3
Make the volume of the cell lysis solution up to 100 ml.
4
The cell lysis solution can now be aliquoted and stored at –20 ºC.
Cell lysis solution (option 1) - contains thiourea.
Reagent
Quantity
Final concentration
Urea (MW 60.06)
42.0 g
7M
Thiourea (MW 76.12)
15.22 g
2M
Tris (1M not pH’d)
3.0 ml
30 mM
CHAPS (MW 614.89)
4g
4% (w/v)
Make up to 100 ml with distilled water
Small aliquots can be stored at -20ºC for up to three months.
Cell lysis solution (option 2)
Reagent
Quantity
Final concentration
Urea (MW 60.06)
48.0 g
8M
Tris (1 M, not pH’d)
3.0 ml
30 mM
CHAPS (MW 614.89)
4g
4% (w/v)
Make up to 100 ml with distilled water
Small aliquots can be stored at -20ºC for up to three months.
2× Sample buffer
For recipes see section 5.5.
2 mM Tris-2-carboxyethyl phosphine hydrochloride (TCEP)
2.8 mg TCEP is dissolved in 5 ml distilled water.
Prepare fresh solution on day of use. TCEP solution is unstable and is to be used
immediately.
Ettan DIGE System User Manual 18-1173-17 Edition AB
39
4 Sample preparation and labeling
4.9 Recipes
40
Ettan DIGE System User Manual 18-1173-17 Edition AB
First dimension isoelectric focusing (IEF) 5
5
First dimension isoelectric focusing (IEF)
5.1
Ettan IPGphor 3 Isoelectric Focusing System
Ettan IPGphor 3 Isoelectric Focusing System and Immobiline DryStrips are
recommended for first dimension electrophoresis, isoelectric focusing (IEF).
A user friendly software, Ettan IPGphor 3 Control software, is provided with the
Ettan IPGphor 3 Isoelectric Focusing System. The software comprises several
features for automatic protocol selection and protocol editing for experimental
monitoring. For documentation purposes it is possible to save log files and
experimental parameters.
The use of a combination of Ettan IPGphor 3 Isoelectric Focusing System and
Immobiline DryStrips generate highly reproducible first dimension isoelectric
focusing results. Up to twelve Immobiline DryStrips can be run at the same time.
Fig 5-1. IPGphor 3 isoelectric focusing apparatus and IPGphor 3 Control software.
Ettan DIGE System User Manual 18-1173-17 Edition AB
41
5 First dimension isoelectric focusing (IEF)
5.2 Workflow
WARNING! The use of Ettan IPGphor Isoelectric Focusing (IEF) System includes
use of high voltage. Read 2D Electrophoresis Principles and Methods, Ettan
IPGphor 3 Instrument User Manual and Ettan IPGphor 3 Safety Handbook prior
to operation of the Ettan IPGphor (IEF) System for detailed instructions and
safety information.
5.1.1
General precautions for good results
2D analysis of protein samples is highly sensitive for contaminations and in order
to achieve good and reproducible results some general precautions must be
considered:
•
Wear gloves to minimize protein contamination.
•
Clean all components with IPGphor Strip Holder cleaning solution (code no.
80-6452-78) and Milli-Q water to remove traces of protein before and after
use.
•
Ensure the Immobiline DryStrips do not dry out.
5.2
Workflow
1
Rehydration of Immobiline DryStrips
2
Sample application to Immobiline DryStrips: Two options described:
3
•
Cup loading protocol
•
Rehydration loading protocol
IEF on Ettan IPGphor 3 apparatus
5.2.1
Sample application protocol selection
The protocol to choose is dependent of the purpose of the experiment, the desired
pH range of the Immobiline DryStrips and sample protein amount and
concentration, see Table 5-1 . For other types of sample application protocols,
see 2D Electrophoresis Principles and Methods handbook.
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Ettan DIGE System User Manual 18-1173-17 Edition AB
First dimension isoelectric focusing (IEF) 5
Table 5-1. Sample application protocol selection criteria.
Experimental conditions
or sample type
Recommended protocol
Samples sensitive for e.g. proteolysis,
protocol minimizes the time the sample
is exposed to room temperature
Cup loading protocol
Samples with volumes up to 150 µl or
with a protein amount under 150 µg.
Large sample loads increase the risk
for protein precipitation at the point of
application
Cup loading protocol
Samples with volumes larger than
100 µl, e.g. diluted samples
Rehydration loading protocol
Experiments using basic Immobilline
DryStrips (pH 6-9, pH 6-11, pH 7-11 NL)
Cup loading protocol or
Samples for preparative
electrophoresis and for large sample
loads (above 150 µg protein)
Rehydration loading protocol or
5.3
Paper-bridge loading protocol (see 2D
Electrophoresis Principles and Methods
handbook for protocol)
Paper-bridge loading protocol (see 2D
Electrophoresis Principles and Methods
handbook for protocol)
Rehydration and sample application
There are two protocols available for rehydration of Immobiline DryStrips and
sample application: Cup loading protocol and Rehydration loading protocol. The
protocols are rather similar. The main difference is that with Cup loading protocol
the samples are added to the strips after rehydration and with Rehydration
loading protocol the samples are added to the strips during rehydration.
Note: Some steps in the protocol below contain two options, one to be used with
the Cup loading protocol and the other with the Rehydration loading
protocol.
5.3.1
Rehydration of Immobiline DryStrips
Immobiline DryStrips must be rehydrated before use. For rehydration an
Immobiline DryStrips Reswelling Tray is used. The Immobiline DryStrips
Reswelling Tray has 12 independent reservoir slots that each hold a single
Immobiline DryStrip. Separate slots allow the rehydration of individual
Immobiline DryStrips in the correct volume of solution.
Ettan DIGE System User Manual 18-1173-17 Edition AB
43
5 First dimension isoelectric focusing (IEF)
5.3 Rehydration and sample application
.
Fig 5-2. The Immobiline DryStrips Reswelling Tray.
Immobiline DryStrips holders can also be used. Information on using Immobiline
DryStrips holders can be found in the 2D Electrophoresis Principles and Methods
handbook.
Protocol
1
Slide the protective lid completely off the Immobiline DryStrips Reswelling
tray. Ensure that the tray is clean and dry. Level the tray by turning the
levelling feet until the bubble in the spirit level is centred.
2
Cup loading protocol: Pipette the appropriate volume of rehydration
solution or DeStreak Rehydration solution into each slot to be used, see
Table 5-2. Deliver the solution slowly along the slot. Remove any large
bubbles. For complete sample uptake, do not apply excess rehydration
solution.
Note: Use DeStreak Rehydration Solution to reduce streaking, especially
in the pH range 7-11. DeStreak Rehydration Solution contains
DeStreak Reagent that prevents unspecific oxidation of protein
thiol groups during electrophoresis.
Rehydration loading protocol: Use labeled protein samples prepared as
described in section, 4.8. The total volume must not exceed the stated
values in Table 5-2 . If the volume is larger the sample must be split or
concentrated. Pipette the appropriate volume of sample into each slot to
be used. Deliver the solution slowly along the slot. Remove any large
bubbles.
44
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First dimension isoelectric focusing (IEF) 5
Table 5-2. Rehydration volumes of Immobiline DryStrips.
Immobiline DryStrips length (cm)
Total volume per strip (µl) including
sample
7
125
11
200
13
250
18
340
24
450
3
Remove the protective cover foil from the Immobiline DryStrips gel. Use
forceps to position the strip with the gel side down. To help coat the entire
strip, 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 DryStrips.
4
Overlay each Immobiline DryStrips with PlusOne™ DryStrips Cover Fluid
to prevent evaporation and urea crystallization.
5
Slide the lid onto the Immobiline DryStrips Reswelling Tray and allow the
Immobiline DryStrips to rehydrate at room temperature. A minimum of 10
h is required for rehydration; overnight is recommended.
Tip! Strips can be rehydrated under low voltage (30 - 50V) when using the
rehydration loading protocol.
5.3.2
Preparations for first dimension run including Cup loading
After rehydration Immobiline DryStrips are prepared for first dimension
isoelectric focusing. Samples are loaded according to the cup loading protocol in
the Manifold.
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45
5 First dimension isoelectric focusing (IEF)
5.3 Rehydration and sample application
Protocol
1
Place precut electrode papers on a clean dry surface such as a glass plate
and soak with deionized water. Remove excess water by blotting with
filter paper.
Note: It is important that the paper electrodes are damp and not wet.
Excess water may cause streaking.
2
Transfer the Immobiline DryStrips from the Reswelling Tray to the
Manifold by using a pair of forceps.
3
Place the Immobiline DryStrips with gel side up and with the acidic end of
the strips oriented toward the anodic side of the instrument.
4
Place a damp paper electrode (from step 1) onto the acidic and basic ends
of the gel.
5
Clip down the electrodes firmly onto the electrode papers. Ensure that
there is good contact between the paper electrodes and the metal.
6
Cup loading protocol only: Clip a loading cup onto the acidic end of the
strip so it is positioned between the two electrodes. The cup should form
a good seal with the Immobiline DryStrips.
Note: To check for a good seal fill the cup to the top with PlusOne
DryStrips Cover Fluid. Observe the level of the fluid to check if it is
decreasing. If a leak is detected remove the PlusOne DryStrips
Cover Fluid and reposition the sample cup.
7
Apply 108 ml of PlusOne DryStrips Cover Fluid allowing the oil to spread
so it completely covers the Immobiline DryStrips (even if fewer than
twelve strips will be loaded in the Manifold).
8
Cup loading protocol only: Add samples prepared as described in
section 4.8. Up to 100 µl of a protein sample can be loaded into the
bottom of the sample cup.
9
Close the lid of Ettan IPGphor 3 instrument.
Now it is possible to start the first dimension isoelectric focusing, see section 5.4.
46
Ettan DIGE System User Manual 18-1173-17 Edition AB
First dimension isoelectric focusing (IEF) 5
5.4
First dimension isoelectric focusing (IEF)
First dimension isoelectric focusing using Ettan IPGphor 3 instrument can be
controlled by either using a PC software (Ettan IPGphor 3 Control software) or the
embedded instrument software.
Use of Ettan IPGphor 3 Control software is recommended for easy and advanced
protocol handling. The software provides a number of optimized protocols for
running first dimension isoelectric focusing. It is also possible to create new
protocols, import existing protocols, and edit protocols. During the run, current
and voltage can be monitored, displayed in a graph, and a log file created. Up to
four different instruments can be monitored from one PC software. See Ettan
IPGphor 3 Control Software User Manual for details.
Protocol (using Ettan IPGphor 3 Control software)
1
Start the Ettan IPGphor 3 Control software and turn on the Ettan IPGphor
3 instrument.
2
Connect the software with the IPGphor 3 instrument on which the run is
to be made.
3
Select protocol for first dimension isoelectric focusing. For selection of
optimized protocols choose Fast mode protocol selection and enter data
of experimental parameters. Alternatively, set protocol using the
Advanced mode protocol selection,
Note: Do not programme the Ettan IPGphor IEF unit to deliver more than
75 µA per Immobiline DryStrips.
4
Start run by clicking the start button in the Ettan IPGphor 3 Control
software. The selected protocol is now downloaded to the selected
instrument and the run is started.
5
If the Immobiline DryStrips are not run immediately on the second
dimension gel, they can be stored at –70°C in a sealed container. The
container has to be rigid because frozen Immobiline DryStrips are very
brittle and can easily be damaged.
Note: Do not equilibrate Immobiline DryStrips prior to storage. This must
be carried out immediately before the second dimension
separation.
Ettan DIGE System User Manual 18-1173-17 Edition AB
47
5 First dimension isoelectric focusing (IEF)
5.5 Recipes
5.5
Recipes
2× Sample buffer/rehydration solution stock (option 1)
Reagent
Quantity
Final concentration
Urea (MW 60.06)
10.5 g
7M
Thiourea (MW 76.12)
3.8 g
2M
CHAPS (MW 614.89)
1g
2% (w/v)
Make up to 25 ml with distilled water
Small aliquots (e.g. 2.5 ml) can be stored at -20 ºC. Stable for 6 months.
2× Sample buffer/rehydration solution stock (option 2)
Reagent
Quantity
Final concentration
Urea (MW 60.06)
12 g
8M
CHAPS (MW 614.89)
1g
2% (w/v)
Make up to 25 ml with distilled water
Small aliquots (e.g. 2.5 ml) can be stored at -20 ºC. Stable for 6 months.
Rehydration solution
48
Reagent
Quantity
2× Sample buffer/
rehydration solution stock 1
or 2
2.5 ml
IPG Buffer, same pH interval
as the Immobiline DryStrips
being rehydrated.
50 µl
DTT
7 mg
Final concentration
2%
Ettan DIGE System User Manual 18-1173-17 Edition AB
First dimension isoelectric focusing (IEF) 5
DeStreak Rehydration Solution
Reagent
Quantity
DeStreak Rehydration
Solution (71-5025-42)
3 ml
IPG Buffer, same pH interval
as the Immobiline DryStrips
being rehydrated
60 µl
Final concentration
2%
DeStreak Rehydration Solution contains DeStreak reagent and optimized
concentrations of Urea, Thiourea and CHAPS.
Note: If IPGphor standard StripHolder or Immobiline DryStrips 7-11NL and
3-11NL are used, use 0.5% IPGphor buffer instead of 2% IPGphor buffer.
2× Sample buffer for CyDye DIGE Fluor minimal dyes
Reagent
Quantity
Final concentration
2× Sample buffer/
rehydration solution stock 1
or 2*
2.5 ml
IPG Buffer, pH 3-10
50 µl
2% (v/v)
DTT (MW 154.2)
50 mg
2% (w/v)
(20mg/ml, 130 mM)
Do not store, prepare fresh before use.
*Use stock option 1 or 2, depending on the rehydration buffer required.
Note: If using DeStreak Rehydration solution, the buffer may contain up to 10 mM
DTT.
2× Sample buffer for CyDye DIGE Fluor saturation dyes
Reagent
Quantity
Final concentration
2× Sample buffer/
rehydration solution stock *
2.5 ml
IPG Buffer, pH 3-10
25 µl
1% (v/v)
DTT (MW 154.2)
5 mg
0.2% (w/v)
(2 mg/ml, 13 mM)
Do not store, prepare fresh before use.
*Use stock option 1 or 2, depending on the rehydration buffer required.
Ettan DIGE System User Manual 18-1173-17 Edition AB
49
5 First dimension isoelectric focusing (IEF)
5.5 Recipes
50
Ettan DIGE System User Manual 18-1173-17 Edition AB
Second dimension SDS PAGE 6
6
Second dimension SDS PAGE
6.1
Ettan DALT electrophoresis system
Ettan DALT electrophoresis systems (Ettan DALTsix and Ettan DALTtwelve) are
recommended for second dimension separation using sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS PAGE). Both systems are designed for
simple assembly and rapid electrophoresis. Ettan DALTsix accepts up to six large
second dimension SDS PAGE gels and Ettan DALTtwelve accepts up to twelve
large second dimension SDS PAGE gels.
The Ettan DALT electrophoresis systems are designed to handle multiple large
format second dimension SDS PAGE gels in a simple, efficient, and reproducible
manner. Ettan DALT gels are large enough to accommodate the longest
Immobiline DryStrips (24 cm).
For Ettan DIGE system applications, low fluorescent glass plates must be used.
These glass plates reduce background signal and will therefore improve the
quality of results.
Ettan DALT gels are poured using a gel caster. Up to 14 Ettan DALT gels can be
prepared in a single batch.
For detailed description of Ettan DALT electrophoresis systems (Ettan DALTsix and
Ettan DALTtwelve), components for preparation of Ettan DALT gels, and for
detailed protocols for loading gels into the caster, and casting Ettan DALT gels
consult the Ettan DALTtwelve system user manual and the Ettan DALTsix system
user manual.
Fig 6-1. Ettan DALTsix (left) and Ettan DALTtwelve (right) electrophoresis systems.
Ettan DIGE System User Manual 18-1173-17 Edition AB
51
6 Second dimension SDS PAGE
6.2 Workflow
6.2
Workflow
1
Cast homogeneous second dimension SDS PAGE gels.
2
Equilibration of focused Immobiline DryStrips.
3
Loading of focused Immobiline DryStrips.
4
Second dimension SDS PAGE.
6.3
Casting homogeneous gels
Note: Prepare gels at least one day before use to ensure reproducible results.
1
Use low fluorescence glass plates without scratches in order to achieve
high quality images with low background.
2
Ensure the entire gel casting system is clean, dry, and free of any
polymerized acrylamide.
3
Prepare 100 ml of displacing solution.
4
Prepare acrylamide gel stock solution. For a full 14-gel set, make up 900
ml of solution. Add TEMED but do not add APS. This amount of gel solution
will provide sufficient volume to cast gels using either a funnel or a
peristaltic pump.
Tip! Use chilled gel solutions. This will increase reproducibility since
temperature effects are minimized.
WARNING! Acrylamide is a neurotoxin. Never pipette by mouth and always
wear protective gloves when working with acrylamide solutions, Immobiline
DryStrips, or surfaces that come into contact with acrylamide solutions.
5
Remove dust by filtering the acrylamide gel stock solution with a
standard bottle top filter apparatus. Dust may create fluorescent
artefactic dots on images.
6
Degas the acrylamide gel stock solution using a vacuum pump.
7
Assemble the gel caster, as described in the Ettan DALT electrophoresis
unit user manuals. The caster should be placed on a level bench or on a
levelling table so that the gel tops are level.
Note: Do not apply excess of filter or separator sheets. Leave 1-2 mm
space from the edge uncovered. Too much pressure will cause gel
distortions.
52
Ettan DIGE System User Manual 18-1173-17 Edition AB
Second dimension SDS PAGE 6
8
Add appropriate volumes of fresh APS to the acrylamide gel stock solution
when ready to pour the gels, and mix thoroughly. Once the APS is added,
polymerization begins.
9
Pour the gel solution until it is about 1-2 cm below the final desired gel
height. Fill the balance chamber with 100 ml of the displacing solution.
The dense displacing solution flows down the connecting tube, filling the
V-well and sloped trough at the bottom of the caster. The remaining
acrylamide solution is forced into the cassettes to the final gel height.
10 Fill the balance chamber with 100 ml of the displacing solution.
11 Spray (do not pipette) overlay solution on the edges of the cassettes, with
for example a plant sprayer, so the edges are covered with a few mm
thick liquid layer using a 0.1% (w/v) spray overlay solution. By doing this
curved edges on the gels are prevented.
Tip! Alternatively, 30% isopropanol can be used to overlay gels.
12 Allow the homogeneous gels to polymerize for at least 3 h before
disassembling the caster. Best polymerization is achieved by letting the
gel polymerize overnight at room temperature.
13 Once gels are completely polymerized cover the top of the gel with SDS
electrophoresis running buffer for the Ettan DALT.
Freshly made gels are best to use. However, gels can be stored in an airtight
container at +4 ºC for up to one week provided they are submersed in 375 mM
Tris-HCl to keep the gels from drying out.
Ettan DIGE System User Manual 18-1173-17 Edition AB
53
6 Second dimension SDS PAGE
6.4 Equilibration of focused Immobiline DryStrips
6.4
Equilibration of focused Immobiline DryStrips
Note: Equilibrate focused strips immediately before the second dimension SDS
PAGE run.
54
1
Prepare SDS equilibration solutions 1 and 2. Allow 10 ml per strip for each
equilibration solution.
2
With forceps carefully remove the Immobiline DryStrips from the IPGphor
Cup Loading Strip Holder. If the Immobiline DryStrips have been focused
and stored frozen, allow the strips to defrost completely beforehand.
3
Place the Immobiline DryStrips in individual equilibration tubes (code no.
80-6467-79) with the support film toward the wall.
4
Add 10 ml of the DTT-containing equilibration solution 1 to each tube.
5
Incubate the strips for 15 min with gentle agitation. Do not overequilibrate, as proteins can diffuse out of the strip during this step.
6
During equilibration, prepare the gel cassettes for loading by rinsing the
top of the gels with running buffer. Place the glass plates in the rack
upside down.
7
For samples labeled with CyDye DIGE fluor minimal dyes: Pour off the
equilibration solution 1 and add 10 ml of equilibration solution 2. Incubate
the strips for 15 min with gentle agitation. Pour off the solution and drain
thoroughly.
8
For samples labeled with CyDye DIGE fluor saturation dyes: Do NOT
use equilibration solution 2 which is containing iodoaceteamide! Use
equilibration solution 1, which contains DTT, instead.
Ettan DIGE System User Manual 18-1173-17 Edition AB
Second dimension SDS PAGE 6
6.5
Loading of focused Immobiline DryStrips
1
Place the gels in the Ettan DALT cassette rack.
2
Briefly rinse the Immobiline DryStrips by submerging them in a
measuring cylinder containing SDS electrophoresis running buffer for
Ettan DALT.
3
Holding one end of the Immobiline DryStrip with forceps, carefully place
the Immobiline DryStrip in-between the two glass plates of the gel. Using
a thin plastic spacer, push against the plastic backing of the Immobiline
DryStrip (not the gel itself) and slide the strip between the two glass plates
until it comes into contact with the surface of the gel.
Note: The strip should just rest on the surface of the gel. Avoid trapping
air bubbles between strip and the gel and avoid piercing the
second-dimension gel with the strip.
Note: The acidic end of the Immobiline DryStrip should be on the right
side of the gel when the shorter of the two plates is facing the user.
Note: The gel face of the strip must not touch the opposite glass plate.
4
Melt an aliquot of agarose overlay solution in a heating block or boiling
water bath for each Immobiline DryStrips. Allow the agarose to cool
slightly and slowly pipette the molten agarose solution, along the upper
surface of the gel, up to the top of the glass plate. Take care not to
introduce bubbles. Do not allow the agarose to solidify.
5
Once the agarose solution has completely set the gel should be run in the
second dimension as soon as practically possible.
Ettan DIGE System User Manual 18-1173-17 Edition AB
55
6 Second dimension SDS PAGE
6.6 Second dimension SDS PAGE
6.6
1
Second dimension SDS PAGE
Fill the lower buffer tank with SDS electrophoresis running buffer for Ettan
DALT. Turn on the control unit, switch on the pump and set the
temperature to 20ºC.
Note: To achieve good results it is important to use the recommended
electrophoresis running buffer with 0.2% (w/v) SDS.
2
When the running buffer has reached the desired temperature, insert the
loaded gel cassettes with the Immobiline DryStrips in place. Load the unit
from back to front. When all 12 slots are filled, the buffer level should be
slightly below the level of the buffer seal gaskets.
Note: Blank cassette must be inserted into any unoccupied slots.
Alternate gel cassettes with blank cassette inserts to facilitate
handling.
Tip! Gel cassettes and blank cassette inserts slide much more easily into
the unit if they are wet. Use SDS electrophoresis running buffer for
Ettan DALT from a wash bottle to wet the cassettes and inserts as
they are being loaded.
3
Pour SDS electrophoresis running buffer for Ettan DALT into the top of the
buffer tank to the fill line.
4
Program the desired run parameters into the control unit, see Table 6-1
for recommendations.
Table 6-1. Recommended running conditions.
Step
Run duration
Effect per gel
Temperature
1
45 min
2W
20°C
2
4h
17 W
20°C
Note: The recommended running conditions are for runs including twelve
gels and should only be used as guidelines. In runs with fewer gels the
electric effect can be increased (up to a maximum of 20 W per gel)
which will reduce run times.
56
5
Close the lid of the buffer tank and press start/stop to begin
electrophoresis.
6
Run the gel until the bromophenol blue dye front reaches the bottom of
the gel.
Ettan DIGE System User Manual 18-1173-17 Edition AB
Second dimension SDS PAGE 6
7
Scan the gels as soon as possible after the second dimension SDS PAGE
is finished in order to minimize protein diffusion, see chapter 7, Image
acquisition. Keep the gels between the glass plates.
Note: Do not fix the gels before the gels are scanned as this may affect
DeCyder 2D software quantitation of CyDye DIGE Fluor minimal
dye labeled proteins.
8
If you cannot scan the gels immediately, store the gels in SDS
electrophoresis running buffer for Ettan DALT at +4ºC in the dark and keep
the gels moist. However, allow the gels to reach room temperature before
scanning. Gels scanned more than a day after running are likely to show
significant diffusion of the protein spots.
Ettan DIGE System User Manual 18-1173-17 Edition AB
57
6 Second dimension SDS PAGE
6.7 Recipes
6.7
Recipes
Displacing solution
Reagents
Quantity
Final concentration
Tris (1.5 M, pH 8.8))
25 ml
375 mM
Glycerol (87% (v/v))
57.5 ml
50% (v/v)
1% Bromophenol blue stock
solution
200 µl
0.002% (w/v)
Make up to 100 ml with distilled water
Prepare fresh and use immediately. Do not store.
12.5% Second dimension SDS PAGE gel composition for Ettan DALT
Reagents
Quantity for 900 ml of a 12.5% gel
Acrylamide/PAGE 40% (w/v)
281.25 ml
PlusOne Methylenebisacrylamide
2% (w/v)
150.3 ml
Tris (1.5 M, pH 8.8)
225 ml
10% (w/v) SDS
9.0 ml
10% (v/v) TEMED
1.24 ml
10% (w/v) APS
9.0 ml
Make up to 900 ml with distilled water
58
•
Use chilled solutions
•
Prior to addition of APS, filter the solution through a 0.2 micron filter into a
clean bottle and then degas the solution
•
Prepare fresh APS solution on day of use
•
Add the APS solution immediately prior to use to prevent polymerization in
the bottle
Ettan DIGE System User Manual 18-1173-17 Edition AB
Second dimension SDS PAGE 6
SDS electrophoresis running buffer for Ettan DALT
Reagents
Quantity
Final concentration
Tris (MW 121.14)
60.5 g
25 mM
Glycine (MW 75.07)
288 g
192 mM
SDS (MW 288.38)
40 g
0.2% (w/v)
Make up to 20 l with distilled water
Store at room temperature. Stable for 3 months.
SDS Equilibration stock solution
Reagents
Quantity
Final concentration
Tris (1.5 M, pH 8.8)
10 ml
75 mM
Urea (MW 60.06)
72.07 g
6M
Glycerol (87% [v/v], MW
92.09)
69 ml
30% (v/v)
SDS (MW 288.33)
4g
2% (w/v)
Make up to 200 ml with distilled water
This stock solution can be stored at room temperature. Stable for 6 months. Add
DTT or Iodoacetamide for equilibration solution 1 or 2.
Equilibration solution 1
Reagent
Quantity
Final concentration
SDS equilibration buffer
stock solution
100 ml
–
DTT (MW 154.2)
0.5 g
0.5% (w/v)
Solution should be used immediately. Do not store.
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59
6 Second dimension SDS PAGE
6.7 Recipes
Equilibration solution 2
Note: Iodoacetamide should not be used in combination with saturation labeling.
Iodoacetamide allows effective alkylation of thiols while minimizing
reoxidation of the competing thiol pairs in protein samples.
Reagent
Quantity
Final concentration
SDS equilibration buffer
stock solution
100 ml
–
Iodoacetamide (MW 185.0)
4.5 g
4.5% (w/v)
Solution should be used immediately. Do not store.
0.5% (w/v) Agarose overlay solution
Reagent
Quantity
Final concentration
SDS electrophoresis running
buffer for Ettan DALT
100 ml
-
Low melting point agarose
prep
0.5 g
0.5% (w/v)
1% Bromophenol blue stock
solution
200 µl
0.002% (w/v)
Mix components in a 250 ml conical flask and heat on a low setting in the
microwave for 1 minute. Ensure all the agarose has melted. Allow the solution to
cool slightly before use. Store at room temperature. Do not keep for more than 1
month.
1% (w/v) Bromophenol blue stock solution
Reagent
Quantity
Final concentration
Bromophenol blue
100 mg
1%
Tris-base
60 mg
50 mM
Make up to 10 ml with double distilled water
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Image acquisition 7
7
Image acquisition
Typhoon Variable Mode Imager is recommended for scanning of DIGE second
dimension SDS PAGE gels. It is also possible to use Ettan DIGE Imager, see section
7.7.
7.1
Typhoon Variable Mode Imager
WARNING! It is important that you read the safety instructions in the Typhoon
User Guide before you start work.
Typhoon Variable Mode Imager is fully optimized as part of Ettan DIGE System
(Fig 7-1). It is optimized to image the CyDye DIGE Fluor dyes characteristics.
Several models of the Typhoon Variable Mode Imager system are available. For
Ettan DIGE system use, Typhoon Variable Mode Imager Trio, 9400 and 9410 are
recommended, as all three CyDye DIGE Fluor minimal dyes can be detected.
However, seamless integration with DeCyder 2D software is ensured for all
models.
Fig 7-1. Typhoon 9410 Variable Mode Imager.
For a detailed guide on Typhoon Variable Mode Imager, see Typhoon User Guide.
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7 Image acquisition
7.2 Workflow
7.2
Workflow
The workflow for scanning gels with Typhoon Variable Mode Imager and
cropping gel image files consist of following steps:
1
Cleaning Typhoon
2
Placing the gels in Typhoon
3
Selecting scan parameters
4
Scanning of the gels
5
Cropping of image files
7.3
Cleaning Typhoon
The following cleaning procedure has been shown to be compatible with the
Typhoon Variable Mode Imager to remove contamination caused by fluorescent
products.
1
Wipe the glass platen with 10% H202 (hydrogen peroxide) using lint free
paper, such as Crew Wipers.
2
Rinse the platen with high purity water. A pre-scan can be done to check
for contaminants that may affect results of scans.
7.4
Placing gels in Typhoon
Note: Wear powder free gloves. The powder used in laboratory gloves can
fluoresce and may also scatter light affecting image quality.
1
Turn on the Typhoon Variable Mode Imager and leave the instrument to
warm up for at least 30 min prior to scanning. Once the instrument is
warmed up it will display READY status.
2
Ensure that the gel glass plates are clean, dry and free from lint.
Tip! For applications using Ettan DIGE system, the recommended glass
plates have low fluorescence characteristics. The gel can then be
scanned still assembled within the plates. Manipulation will be easier
and there will be less risk of damage to the gel.
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Image acquisition 7
3
Position the main bar of the Gel Alignment Guide onto the platen.
grippers
front location bar
4
Using the grippers, position the dried glass plate assembly with one edge
on the spacer and against the front location bar and gently lower the
assembled gel onto the platen.
5
Position the gel by using the Gel orientation guide. The physical gel
orientation should be noted by the user, the gel orientation option in the
software determines the file output orientation.
Basic
Acidic
High
Mw
Low
Mw
Tip! Using Ettan DALT trays, it is recommended that Ettan DALT gels are
scanned with the short glass plate facing down to the platen.
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7 Image acquisition
7.5 Scan parameters and scanning
6
7.5
Close the instrument lid.
Scan parameters and scanning
Start the Typhoon Scanner Control software. The Typhoon Scanner Control Multiple Sample scan window is opened (Fig 7-2).
Fig 7-2. The Typhoon Scanner Control - Multiple Sample scan window.
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Image acquisition 7
1
Select scan area (pre-defined tray area) within the Tray set-up area
(Labeled 1 in Fig 7-2), use the drop down window to select the appropriate
predefined tray, i.e., the DIGE Ettan DALT. In this mode, the scan area is
pre-defined and the software is able to recognize where individual gels
will be located, resulting in separate file outputs for each gel.
Tip! The Tray Editor can be used to create tray areas for gel types other
than Ettan DALT. In User Select mode, only the scan area is predefined. The software is unable to recognize where individual gels
will be located, therefore a single file output will be generated.
Manual cropping is required following the scan.
Define the number of gels to be scanned. Use the drop down menu in the
Tray set-up area (labeled 1 in Fig 7-2). In the DIGE Ettan DALT mode up to
two gels can be selected.
2
Use the drop down menu under the Acquisition Mode heading (labeled 2
in Fig 7-2) to select fluorescence as acquisitor mode.
Note: Scan modes other than fluorescence are available, these are
covered in the Typhoon User Guide.
3
Set up fluorescence scan parameters:
a
Click the Setup button (Labeled 3 in Fig 7-2) to activate the
Fluorescence Setup window.
b
Select the number of scan channels to be programmed for the sample
on the platen. Between one and four channels can be programmed.
To select or deselect scan channels, click the Use check box.
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7 Image acquisition
7.5 Scan parameters and scanning
c
Select appropriate emission filters from the Emission Filter list. This list
displays the filters that are installed on the Typhoon Variable Mode
Imager along with a description of the typical filter use. The Scanner
Control software automatically suggests the laser to use with the
emission filter selected. See Table 7-1 for recommended emission
filters and laser combinations. The CyDye DIGE Fluor dye filter and
laser combinations are selected to give the optimum results with
minimal cross-talk.
Table 7-1. Recommended emission filters and laser combinations.
Dye
Emission Filter (nm)
Laser
Cy2
520 BP 40
Blue2 (488)
Cy3
580 BP 30
Green (532)
Cy5
670 BP 30
Red (633)
Deep Purple™
560 LP
Green (532)
Tip! For Deep Purple™-post-stained analytical gels it is recommended to use a
457 nm laser excitation in conjunction with a 610 nm band pass emission
filter (or equivalent if not using a Typhoon scanner). This will minimize any
potential cross-talk between Deep Purple and the CyDye DIGE fluor dyes.
66
d
Set the PMT voltage for each scan wavelength. A quick pre-scan at
500 or 1 000 µm pixel resolution should be performed initially to
identify a suitable voltage. For further details, see section 7.5.1.
e
Select normal as Sensitivity scan setting for each scan wavelength
(usually sufficiently sensitive for 2D DIGE applications).
f
Select the Sensitivity check box. It is essential that this is selected for
analytical gels to ensure that all scans are carried out as individual
scans.
g
Click OK.
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Image acquisition 7
4
Select orientation (orienting options button labeled 4 in Fig 7-2), see
section 7.4 step 5.
Tip! To aid selection of the correct character the Gel Orientation Guide
can be overlaid above the gel once it is in position on the Typhoon
platen. The appearance of the overlaid letter “R” on the Gel
Orientation Guide indicates which character to select on the
Typhoon Scanner Control software screen.
Note: ImageQuant™ TL Tools can be used to flip or rotate images which
were scanned in wrong orientation.
5
Select the Press Sample option (labeled 5 in Fig 7-2) if scanning gels
between glass plates.
CAUTION! The Press Sample feature should not be used with naked or
backed gels as it can damage the instrument.
6
7
Select Pixel size from the list (labeled 6 in Fig 7-2). A quick pre-scan at
1000 µm pixel resolution should be performed initially to identify a
suitable PMT voltage.
•
Select 100 µm as pixel size for DALT gels.
•
Select 25 or 50 µm as pixel size for minigels.
Select Focal Plane from the list (labeled 7 in Fig 7-2). The +3 mm setting is
used for most applications of the Ettan DIGE system (assembled gels or
samples held on a glass plate).
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7 Image acquisition
7.5 Scan parameters and scanning
8
Make sure the instrument lid is shut. Select DIGE file naming format. Check
the DIGE File Naming Format box (labeled 8 in Fig 7-2) in the Scanner
Control window to ensure that unique filenames can be generated for
each scan channel.
Tip! Using the DIGE File Naming Format option results in all files having
user defined filenames. All scan images from a given experiment can
be saved into a single user defined folder. This method of file naming
and folder selection results in structures that can be directly used to
crop image for subsequent DeCyder 2D analysis.
9
Start the scan by clicking the Scan button (labeled 9 in Fig 7-2) in the
Scanner Control window. The DIGE File Naming Format window appears.
•
If a single scan setting has been chosen, e.g. for a Deep Purple stained
gel, then the resulting output on scan completion will be a filename.gel
file in the selected folder.
•
If two or more scan parameter settings were chosen, e.g. for a
Cy2/Cy3/Cy5 gel, then the resulting output upon scan completion will
be a filename.ds file in the selected folder and a new folder called
filename.dir. In this new folder will be the user named.gel files. The
filename.ds file allows the scanned images to be overlaid in
ImageQuant TL whilst the user named.gel files are the individual scan
channel outputs and can be viewed as separate files.
10 The terms STANDARD, Cy2, Cy3 and Cy5 are automatically appended to
the file. These terms are also automatically picked up during image
analysis so reducing the requirement for user input.
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Image acquisition 7
11 The term STANDARD can be assigned to the file name of the image which
contains the internal standard by clicking the relevant check box.
If not required the STANDARD name can be removed by clicking the NONE
check box. The filenames can also be manually edited.
12 Click the OK button. The Multiple Sample Name dialog is displayed.
13 Save the images by choosing the folder and filename for each gel
individually. Click on Edit Sample File Name...
14 Entering details using Folder or Base File Name, allows the user to Set
common details for all gels in a single operation. The Browse option in
both cases allows the user to select existing folders or file name
structures. A number of gels can be set up for a single scan run and obtain
unique filenames for each gel image.
15 Click SCAN to start the scan. Once the scan has started, the preview
window appears.
•
For unlinked scans a single image channel appears for each scan
programmed, the images appearing one at a time.
•
For linked scans, two image channels appear simultaneously.
•
Where more than one gel is scanned using the DIGE Ettan DALT tray
settings, a drop down numerical menu appears allowing the user to
monitor each of the gels as the images are generated.
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7 Image acquisition
7.5 Scan parameters and scanning
16 Evaluate the scan progress. Saturated data is displayed in red in the
preview window. If saturated data is seen the user can cancel the scan
and re-set the PMT voltage without having to complete the whole scan.
17 Handle image file output. The image files created are labeled as
filename.gel. This uses a modified 16 bit TIF format. An additional text file,
filename.ds, also exists and this links image file data together for image
overlays in ImageQuant TL.
7.5.1
Pre-scanning to identify a suitable PMT voltage
The PMT voltage can be set from 300 to 1 000 V although it is recommended that,
where possible, work is performed between 400 and 900 V. The voltage chosen
depends on the type and quantity of dye or stain present. A quick prescan at 500
or 1 000 µm pixel resolution should be performed to identify a suitable voltage.
This allows a rapid scan at a relatively low resolution that should not be used for
quantitative analysis. It does however give an approximation of expected signal
values which will aid determination of the PMT voltage required. The prescan can
be opened in ImageQuant TL software. Spots showing the most intense signal
should be selected using one of the Object tools such as the rectangle.
Higher resolution 100 µm scans must be used to collect quantitative data. This
resolution is required for subsequent data analysis using DeCyder 2D.
Using the Volume Review tool button displays the information associated with
the selected area in the format.
Note: The maximum pixel value should not exceed 100,000 as this indicates
signal saturation has been reached and this will prevent quantitative
analysis being achieved.
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Image acquisition 7
A target maximum pixel value of 50 000 to 80 000 is usually suitable. When
adjusting the voltage, relatively small increments of 20 to 50 volts are
recommended. If only one or two spots show saturation then only slight
downward adjustments to the PMT voltage setting are normally required. Once
the voltage has been optimized for one gel in an experiment, these settings can
be used for all similar gels within the same experiment. The maximum pixel value
should be within the specified range for all gels, to enable accurate quantitation
of spot volumes.
7.6
Cropping using ImageQuant TL
Prior to image analysis the image files should be cropped to exclude nonessential
information from the image files. To exclude the nonessential information,
ImageQuant TL Tools should be used to crop the images prior to image analysis.
Further cropping of the individual gel areas is normally required to remove
supplementary data and can be performed in ImageQuant TL Tools.
1
Define an area of interest within ImageQuant TL Tools using the dashed
square button or use the “Tools” menu.
2
Crop using Edit:Crop Dataset or the crop current dataset button on the
toolbar.
3
For image analysis, save the cropped images by selecting File:Export Gel
Files from Dataset to Folder. This method only saves the name.gel files
and allows images from multiple gels to be saved in a common folder.
4
To retain dataset functionality, save the cropped images by selecting:
File:Save As….
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7 Image acquisition
7.6 Cropping using ImageQuant TL
7.6.1
ImageQuant TL trouble shooting
If out of memory messages appear while using ImageQuant TL, causing the
software to crash, this may be corrected by decreasing the virtual memory under
1Gb. Remember to change the setting again when using the image analysis
softwares, which need a large virtual memory to function correctly.
To set the size of the virtual memory:
72
1
Right-click on My computer and select Properties.
2
Select the Advanced tab.
3
In the Performance area, click the Settings... button.
4
In the Performance area, click the Settings... button.
5
Click Change... to change the virtual memory to the appropriate value
and click OK to change the setting.
6
Restart the computer to apply the new setting.
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Image acquisition 7
7.7
Ettan DIGE Imager
Ettan DIGE Imager is a scanning CCD camera-based instrument designed for high
resolution images of 2D DIGE applications (Fig 7-3). In particular it can create high
quality images of 2D DIGE gels. By combining very high resolution with precise
motion control, the Ettan DIGE Imager produces accurate multi-channel images
of Cy2, Cy3 and Cy5 labeled gels. The system has also been designed to image a
wide range of other fluorescent gel and membrane applications.
Fig 7-3. Ettan DIGE Imager.
The imager is controlled using Ettan DIGE Imager software, and can be set up for
a variety of gel and membrane formats. Data produced by Ettan DIGE Imager is
directly compatible with ImageQuant TL, ImageMaster 2D Platinum and DeCyder
2D. For information about how to use the imager, refer to Ettan DIGE Imager User
Manual.
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7 Image acquisition
7.7 Ettan DIGE Imager
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Image analysis 8
8
Image analysis
Two different softwares have been specially designed for image analysis in Ettan
DIGE System: DeCyder 2D and ImageMaster 2D Platinum software. DeCyder 2D
is recommended in complex experiments when more than two groups or
conditions are used in an experiment. ImageMaster 2D Platinum software is
suitable for basic experiments containing control vs. treated or healthy vs. nonhealthy conditions.
These dedicated 2D software products use the internal standard to minimize gelto-gel result variation. A detection of less than 10% difference between samples
can be made with more than 95% statistical confidence.
Tip! Ensure DIGE images are cropped before being loaded into DeCyder 2D.
8.1
DeCyder 2D software
DeCyder 2D software is specially designed for Ettan DIGE system use. It enables
the production of quantitative data of unparalleled accuracy, supported by
statistical tests. This gives confidence that the results achieved reflect true
biological outcomes and are not due to the system. DeCyder 2D is a fully
automated image analysis software suite for detection, quantitation, positional
matching and differential protein abundance analysis. An optional add-on
module Extended Data Analysis (EDA) can handle up to 1000 spot maps. The raw
data (gel images) are linked to EDA and multivariate analysis of data can be
opened for display via the BVA module.
This section briefly outlines the features and capabilities of the software. For a
detailed guide, please refer to the DeCyder 2D Software User Manual and
DeCyder Extended Data Analysis module User Manual which both include a
series of tutorials designed to provide new users with the means to gain a rapid
understanding of the software’s capabilities. Online helps are also integrated with
the software, providing help for the different parts of the software.
8.1.1
Modules
DeCyder 2D version 6.5 software comprises six modules (where the EDA module
is optional):
Image Loader
Import of scanned gel images into a project within the DeCyder database, making
them accessible for other modules. The import must be performed before
analyses can be performed in DeCyder 2D software.
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8 Image analysis
8.1 DeCyder 2D software
Batch Processor
Fully automated image detection and matching of multiple gels without user
intervention. Proteins of interest can be automatically filtered and picking list
generated.
DIA (Differential In-gel Analysis)
Protein spot detection and quantitation on a set of images, from the same gel.
Features include background subtraction, in-gel normalization and gel artefact
removal. Images must be processed in the DIA interface prior to data analysis in
BVA.
BVA (Biological Variation Analysis)
Matching of multiple images from different gels to provide statistical data on
differential protein abundance levels between multiple groups.
XML Toolbox
Extraction of user specific data from XML files generated in either the Batch, DIA
or BVA modules. This data can be saved in either text or html format enabling
users to access data from DeCyder 2D workspaces in other applications.
EDA (Extended Data Analysis)
Multivariate analysis of data from several BVA workspaces. EDA is an add-on
module for the DeCyder 2D software and can handle up to 1000 spot maps. The
raw data (gel images) are linked to EDA and can be opened for display via the BVA
module. In addition to the univariate analyses that can be performed in the BVA
module, it is also possible to perform the following analyses in EDA:
76
•
Principal Component Analysis: Produces an overview of the data. Can
be used to find outliers in the data.
•
Pattern analysis: Finds patterns in expression data (e.g. proteins and
spot maps with similar expression profiles).
•
Discriminant analysis: Finds proteins that discriminate between
different samples (to find biological markers), creates classifiers and
assigns samples to known classes depending on expression profiles (e.g.
tumor typing).
•
Interpretation: Finds the biological context of proteins by integrating
biological information and context from in-house or public databases. It
can be used to determine in what pathways and processes a protein is
involved, the function of the protein etc.
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Image analysis 8
8.2
ImageMaster 2D Platinum software
8.2.1
Structure
ImageMaster 2D Platinum ensures fast and reliable image comparisons. It easily
manages multiple image analyses and offers the possibility to automate
detection and matching steps with numerous interactive tools for optimizing and
manipulating data. Furthermore, it is possible to attain a higher level of
quantitative and qualitative analysis using the robust and sophisticated
techniques provided in the application. ImageMaster integrates filtering,
querying, reporting, and statistical and graphing options so that you can easily
view, compare, analyze and present your results.
The powerful suite of features that normally apply to 2D gels in ImageMaster can
now be used in conjunction with DIGE gels by using the ImageMaster 2D Platinum
6.0 DIGE module of the software.
This section briefly outlines the features and capabilities of the software. For a
detailed guide, please refer to the ImageMaster 2D Platinum User Manual which
also includes a series of tutorials designed to provide new users with the means
to gain a rapid understanding of the software’s capabilities. An online help is also
integrated with the software.
8.2.2
Image analysis workflow
A typical image analysis using ImageMaster 2D Platinum software for DIGE gels
would consist of the steps below. For detailed information, see the ImageMaster
2D Platinum User Manual.
1
Acquiring data
Gel images must first be digitized using an image capture device. This will
generally be done with a separate software. Open gels from TWAIN
compatible scanners with ImageMaster.
2
Setting up a workspace
Set up a workspace to open and work on gel images. The workspace allows
to organize gels into projects, to define match sets and classes and to keep
accompanying data, such as reports and image documents in project
related folders. Preferred ImageMaster settings are also saved in the
workspace file.
3
Visualizing gels
This step is to handle the gel files (open, save, print, close), manipulate the gel
images (select, move, zoom, stack, align), possibly transform the gels (rotate,
crop, scale) and view the signal intensity (adjust contrast, profile, 3D view).
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8 Image analysis
8.2 ImageMaster 2D Platinum software
4
Detecting and quantifying spots
Perform automatic spot detection. It is also possible to select, display and
edit spots, as well as view spot properties and quantification values.
5
Annotating spots and pixels
Individual pixels and spots in a gel image may be labeled with annotations.
These annotations can be used in functionalities such as calibration,
alignment and matching, or be utilized to mark spots with their particular
characteristics. Create, use, select and display labels, categories and
annotations. It is also possible to create links to external databases or data
sources of any format (text, file, html, etc.).
6
Matching gels
After spots were detected and match sets defined gel images can be
matched.
7
Analyzing data
Data analysis and classifications tools to study the variations in protein
expression among gels or classes of gels can be performed. The data
analysis step may be carried out at two different levels. The intra-class
statistics tools include scatter plots, descriptive statistics, and factor
analysis. For inter-class analyses, the so-called overlapping measures and
various statistical tests can be used. Heuristic clustering can help finding
classes.
8
Integrating data
Exports spot coordinates to a spot excision robot, exports gel data to a
database (for example, via XML format), or imports experimental information
to be included in annotations.
9
Reporting results
Display information on specific gels and gel components (spots, matches,
classes, annotations) at any moment during the analysis. It is possible to
display, use, save, customize and edit reports. Several specific report types
are available.
10 Controlling and automating gel analyses
As with reporting results, operations that were carried out on gels can be
checked at any time using the History function. It is also possible to create
Scripts for automating parts of your analysis. A multiple undo/redo feature is
also available.
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Preparative workflow 9
9
Preparative workflow
9.1
Introduction
When proteins of interest have been identified in 2D DIGE experiments the
corresponding spots can be picked and analyzed in a mass spectrometer for
protein identification. Although spots of interest can be picked directly from poststained analytical SDS PAGE gels, spot picking from preparative-scale gels
provide more material for analysis by mass spectrometry.
Ettan DIGE system is fully compatible with mass spectrometry analysis:
1
A preparative gel can be matched to previously run analytical gels by
DeCyder 2D software.
2
DeCyder 2D software will generate a pick list of spots of interest that can be
directly exported to Ettan Spot Picker or Ettan Spot Handling Workstation.
3
Protein spots of interest are then automatically picked from the gel by Ettan
Spot Picker or Ettan Spot Handling Workstation.
This chapter provides information of changes to the protocols described in
chapters 4-8 one must make to ensure an efficient preparative workflow. The
workflow is outlined in Fig 9-1.
9.1.1
Staining of preparative gels
Depending on which CyDye DIGE Fluor dye has been used for sample labeling
there are two options for visualization of preparative gels:
•
For CyDye DIGE Fluor minimal dyes, the second dimension SDS PAGE
preparative gels must be post stained. Use fluorescent protein stains, such
as Deep Purple, for post-staining.
Note: CyDye labeled proteins are approximately 450 Da larger in molecular
weight compared to non-labeled proteins.
•
For CyDye DIGE Fluor saturation dyes no post-staining is required. Use the
preparative dye vial provided in the kit instead. This vial provides amounts of
CyDye DIGE Fluor saturation dyes enough for labeling preparative samples.
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9 Preparative workflow
9.1 Introduction
1. Sample preparation and labeling
Unlabeled
Cy3
Alternative 1 - Unlabeled sample, post-staining required
Protein extraction
Cells
Sample
Alternative 2 - Labeled sample, no post-staining required
Labeling with CyDye DIGE Fluor
saturation dyes (preparative vial)
Protein extraction
Sample
Cells
2. Perform first dimension electrophoresis
Ettan IPGphor 3 Isoelectric Focusing System
3. Prepare the Ettan DALT gel
Bind-Silane treatment
Attach reference markers
Cast the gel
Glass plate
4. Perform second dimension electrophoresis
Ettan DALTtwelve (or Ettan DALTsix) electrophoresis system
5. Post-stain and scan the gel
Scan the gel
Stain the gel
Preparative gel
Post-stained gel
Scanned gel image
6. Match the gels and create a pick list
Matching gels
Creating a pick list
Matching
Preparative
gel image
Master image
Proteins of interest
found in the analytical
workflow
Pick list
+
.txt
Pick gel
(stained preparative gel)
7. Spot pick the post-stained gel
Pick list
MALDI slides
+
.txt
Pick gel
(stained preparative gel)
Ettan Spot Picker
8. Perform mass spectrometry analysis
Mass spectrum
MALDI slides
Protein ID
M/Z
Fig 9-1. Preparative gel workflow in Ettan DIGE System.
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Preparative workflow 9
9.2
Sample preparation
Amount of sample
Prepare samples as described in chapter 4. However, on a preparative gel larger
amounts of protein sample are usually loaded, typically 500 µg or more.
Protein labeling
Preparative gel samples do not require labeling with CyDye DIGE Fluor minimal
dyes. It is recommended to use Deep Purple for post-staining instead, see section
9.5.
However, if samples have been labeled with CyDye DIGE Fluor saturation dyes
they can be used directly in preparative gels. In CyDye DIGE Fluor Labeling Kit for
Scarce Samples and Preparative Gel Labeling a vial of Cy3 dye is included which
allows labeling of up to 500 µg of protein following the protocol 25-8009-83PL.
9.3
First dimension isoelectric focusing (IEF)
For preparative workflow first dimension isoelectric focusing follow protocols in
chapter 5 considering:
•
Use rehydration loading (section 5.3) or paper-bridge loading (see 2D
Electrophoresis Principles and Methods handbook) protocols for sample
application.
•
The loading should be optimized for different strip lengths and pH ranges.
For a pH 3-10 NL 24 cm Immobiline DryStrips, up to 600 µg of protein can be
loaded on a preparative gel.
9.4
Second dimension SDS PAGE
For preparative workflow second dimension SDS PAGE electrophoresis please
consider:
•
Clean and Bind-Silane treat the glass plates to be used
•
Attach reference markers to the treated glass plates
•
Ensure correct orientation of Immobiline DryStrips and the preparative
second dimension SDS PAGE gel
9.4.1
Clean and Bind-Silane treat the glass plates
The Bind-Silane treatment of the glass plates is performed to immobilize the gel
onto the glass plate and to prevent the gel from deforming during the staining,
imaging and picking processes.
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9 Preparative workflow
9.4 Second dimension SDS PAGE
The following protocol for treatment of glass plates was optimized for PlusOne
Bind-Silane.
Note: Use the shorter glass plate without reference markers.
1
Thoroughly scrape off any residual bound gel with a plastic scraper and
wash the plate in 1% Decon™ (v/v) (branded Contrad™ in the USA) with a
soft sponge to further remove the gel.
2
Leave the plate to soak in 1% Decon (v/v) overnight. On the following day,
wash the plate with a soft sponge.
3
Rinse the plate with water and leave the plate to soak in 1% HCl (v/v) for
1 h.
4
Wash the plate in 1% Decon (v/v) with a soft sponge, then rinse with
double distilled water.
5
Dry the plate using a lint-free tissue or leave to air dry in a dust free
environment. If not to be used immediately, please store in a dust free
environment.
6
Prepare the Bind-Silane working solution.
7
Pipette 2-4 ml (depending on plate size) of the above solution over the
whole surface of the plate and wipe it over with a lint-free tissue until it is
dry. Cover the plate with a lint-free tissue to prevent dust contamination
and leave on the bench for 1.5 h (minimum one hour) for excess BindSilane to evaporate.
Note: If the Bind-Silane is not left to dry sufficiently before the glass plates
are assembled for casting, the solution will evaporate off the
treated plate and coat the facing glass surface. This will cause the
gel to stick to both plates when it sets.
Note: The gels will stay attached to Bind-Silane treated glass during
electrophoresis, staining procedures, scanning and storage.
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Preparative workflow 9
9.4.2
Attach reference markers
Reference markers are used to ensure that the correct protein spots detected
during image analysis are picked.
Reference markers must be attached to the short treated glass plate before gel
pouring. It is important that the markers are appropriately placed on the treated
surface of the Bind-Silanized plate. Take care not to place the markers where they
will interfere with the pattern of protein spots in the gel. The markers should be
placed according to the following protocol.
1
Measure the length of the treated plate edge.
2
Place the marker approximately half-way along this edge, away from the
spacer, but not so far as to interfere with the protein spot pattern. The
marker should not touch the spacer. Make sure that the markers are
firmly stuck to the plate by pressing down with a lint free tissue or powder
free glove.
3
Repeat steps 1 and 2 for the other edge of the treated backing plate.
4
When finished, the markers should be in positions similar to those shown
below.
Fig 9-2. Diagram showing the preferred position of reference markers on the gel
backing (short glass plate).
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9 Preparative workflow
9.5 Post-staining with Deep Purple Total Protein Stain
9.4.3
Second dimension SDS PAGE
We recommend using the Ettan DALT electrophoresis system.
The orientations of the Immobiline DryStrips and the reference markers are
critical to ensure that the picking gel is easily matched to the analytical gels
during image analysis, and that the correct spots are picked from the gel when
the pick list is exported to the Ettan Spot Picker.
1
After a picking gel has been poured containing suitable reference
markers, position the gel so the front of the reference markers are facing
you.
2
Equilibrate the Immobiline DryStrips as described in Section 6.4,
Equilibration of focused Immobiline DryStrips.
3
Load the equilibrated Immobiline DryStrips (see Section 6.5, Loading of
focused Immobiline DryStrips), ensuring that the acidic (pointed end) of
the Immobiline DryStrips is on the left hand side of the gel as shown in Fig
9-2.
4
To run the gel, see 6.6 for further information.
9.5
Post-staining with Deep Purple Total Protein Stain
Note: If the sample used has been labeled with CyDye DIGE Fluor saturation dye
post-staining of the second dimension SDS PAGE gel is not required.
The fluorescent Deep Purple Total Protein Stain fits into the standard 2D gel
electrophoresis workflow and is particularly suitable for use with the Ettan DIGE
system. The recommended workflow involves the matching of Deep Purple poststained preparative gels with CyDye labeled analytical gels. Deep Purple has
been shown to be compatible with the image analysis softwares and the stain is
compatible with manual or automated spot picking and mass spectrometry for
protein identification applications.
1
Place an appropriate volume of fixation solution 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 (1000 ml for Ettan
DALT gels).
2
Dismantle the electrophoresis apparatus. Remove one glass plate and
place the gel attached to the glass plate into fix solution.
Note: Place only one gel in each container. The Immobiline DryStrip can
be left attached to help with gel orientation.
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Preparative workflow 9
3
Incubate in the fixation solution overnight at room temperature with
gentle agitation.
4
Take the stain out of the -15°C to -30°C freezer and allow to stand at room
temperature for 5-10 minutes.
5
Pour off the fixation solution and replace with the wash solution (1000 ml
for Ettan DALT gels). Wash with gentle agitation for 30 minutes.
6
Pour off the wash solution and replace with 500 ml water. To make up the
working stain solution, briefly shake the stain concentrate and add 2.5 ml
Deep Purple to make a 1:200 dilution. Cover the container to create a dark
environment and incubate for 1 hour at room temperature with gentle
agitation.
Note: The solution is light sensitive and should be kept out of bright light.
Note: Containers can be wrapped in foil or covered with black plastic. It is
not necessary to eliminate light completely, only to ensure that
bright light is significantly reduced. Alternatively, containers with
lids, that are a solid colored plastic, may be used.
7
Pour off the stain and replace with 7.5% (v/v) acetic acid. Cover the
container to create a dark environment and incubate with gentle
agitation for at least 15 minutes.
8
Repeat the acetic acid step once. The gel can be imaged at this stage.
Note: If speed is more important than background levels, the gel can be
imaged after one acetic acid step. Further washes in acetic acid can
be performed to reduce the background still further if necessary.
After imaging, the gels can be stored in the dark in 7.5% (v/v) acetic
acid at 2–8 ºC for several weeks. This allows for further imaging at
a later date if required.
9.6
Gel scanning
We recommend using Typhoon Variable Mode Imager or Ettan DIGE Imager.
Please refer to Chapter 7 for more details.
1
Place the gel (glass side down) onto a clean, dust-free platen surface.
2
Image the gel with the appropriate filter set and exposure times. It is
recommended that the image resolution for the analytical and
preparative gels are set at the same level and are at least 100 µm.
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9 Preparative workflow
9.7 Matching gels and creating a pick list
3
Ensure that both reference markers can be clearly seen and that they
appear as circles when the gel image is checked. If the markers cannot be
seen, then re-scan the gel, adjusting the area to be scanned
appropriately.
4
When scanning is finished, place the gel into the storage solution (7.5%
acetic acid) at 4°C in the dark.
5
Clean the scanner platen after use to remove any fluorescent residue. If
the platen is not thoroughly cleaned, this residue can interfere with
subsequent scans producing high background levels. See 7.3 for
information of how to clean the scanner platen.
9.7
Matching gels and creating a pick list
The matching of gels and creating a pick list is performed in DeCyder 2D. For
information on the different steps in the workflow below, see DeCyder 2D User
Manual and DeCyder Extended Analysis module User Manual.
9.8
Spot picking the gel
After a pick list has been created from the image analysis software, spot picking
can be performed. The procedure of picking and digesting spots can be
performed by manual transfer of gels and microplates between the Ettan Spot
Picker, Ettan Digester and Ettan Spotter, or fully automatically in the integrated
Ettan Spot Handling Workstation.
Processing is done in three main steps:
86
1
Spot picking from second dimension SDS PAGE gels
Selected protein spots from stained gels are automatically picked by Ettan
Spot Picker using a pick list created from the image analysis software, and
the gel plugs are transferred into microplates. See Ettan Spot Picker User
Manual.
2
Digestion of the picked proteins
The gel plugs are first trypsin digested in Ettan Digester and the resulting
peptides extracted from the gel plugs. See Ettan Digester User Manual.
3
Spotting of samples onto MALDI targets
The extracted peptides are mixed with matrix solution and spotted on MALDI
targets using Ettan Spotter. See Ettan Spotter User Manual.
Ettan DIGE System User Manual 18-1173-17 Edition AB
Preparative workflow 9
Ettan Spot Handling Workstation automatically processes and transfers
biomolecules from polyacrylamide gels to MS targets. A computer running
proprietary software controls the whole process. For detailed instructions, see
Ettan Spot Handling Workstation User Manual.
9.9
Mass spectrometry analysis
MALDI targets are analyzed by MALDI-ToF MS for protein identification. Time-offlight mass spectrometry is a technique for analyzing molecular weights based
on the motion of ionized samples in an electrical field. In MALDI-ToF, a matrixbound sample is bombarded with a pulsed laser beam to generate ions for
subsequent detection.
Protein identification by mass spectrometry is usually performed on spot-picked
unlabeled protein, visualized on the second dimension SDS PAGE gel with a postelectrophoresis stain, such as Deep Purple. Some applications may require direct
spot picking from a second dimension SDS PAGE gel containing protein labeled
with CyDye DIGE Fluor minimal dye. The nature of the minimal labeling approach
results in the majority of the protein (and peptide) population remaining
unlabeled.
The results can be imported into DeCyder 2D software modules BVA and EDA for
further analysis in the EDA module.
9.10
Recipes
Recipes for post-staining of gels:
Bind-Silane working solution
Reagent
Quantity
Ethanol
16 ml
Glacial acetic acid
400 µl
Bind-Silane
20 µl
Double distilled H2O
3.6 ml
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9 Preparative workflow
9.10 Recipes
Fixation solution
Reagent
Quantity
Final concentration
Ethanol
100 ml
10% (v/v)
Acetic acid
75 ml
7.5% (v/v)
Make up to 1 000 ml with distilled water.
~ 20-fold excess of the gel volume should be used.
Wash solution (small, usually free-floating, gels)
Reagent
Quantity
Final concentration
Na2CO3
21.2 g
200 mM
Dissolve Na2CO3 in 750 ml water.
Make up to 1 000 ml with distilled water.
The pH of the solution should be at least 11 and should be verified.
~ 20-fold excess of the gel volume should be used.
This solution can be stored for up to 2 weeks.
Wash solution (large, usually backed, gels)
Reagent
Quantity
Final concentration
Na2HCO3
2.94 g
35 mM
Na2CO3
31.8 g
300 mM
Dissolve Na2HCO3 and Na2CO3 in 750 ml water.
Make up to 1 000 ml with distilled water.
The pH of the solution should be pH 10-11 and should be verified.
~ 20-fold excess of the gel volume should be used.
This solution can be stored for up to 2 weeks.
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Preparative workflow 9
Working stain solution
Reagent
Quantity
Final concentration
Deep Purple
2.5 ml
1:200 dilution
Make up to 500 ml with distilled water.
~ 10-fold excess of the gel volume should be used.
This solution should be made fresh at the time of use by adding an appropriate
aliquot of Deep Purple to water in the gel staining tank. If necessary it is possible
to store this solution, protected from exposure to light, for up to 1 week at 2-8°C
or 24 h at room temperature.
Storage solution
Reagent
Quantity
Final concentration
Glacial acetic acid
75 ml
7.5% (v/v)
Make up to 1 000 ml with distilled water
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9 Preparative workflow
9.10 Recipes
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Testing cell lysates for successful labeling A
Appendix A
Testing cell lysates for successful labeling
A.1
Testing new cell lysate for successful labeling
It is important to check that labeling of the proteins is optimized before the
samples are taken through the 2D electrophoresis process.
The method involves running a small sample of the freshly labeled lysate on a 1D
SDS PAGE gel along with a control lysate already known to label successfully. The
gel is then scanned at the appropriate wavelength for the relevant CyDye DIGE
Fluor dyes. The total fluorescence of each labeled sample is then compared. The
method should also be used to test protein lysates that contain previously
untested chemical components.
Protocol
1
Label 50 µg of the new protein sample with 400 pmol of CyDye DIGE Fluor
Cy5 minimal dye.
2
Add a volume of each labeled protein lysate equivalent to 50 µg, to a
microfuge tube.
3
Add an equal volume of the 2× gel loading buffer to the labeled protein
lysate.
4
Heat the samples at 95 ºC for 5 min to ensure full reduction of the
proteins.
5
Make a serial dilution of each of the lysates in the 2× gel loading buffer,
e.g. 25 µg, 12.5 µg and 6.25 µg.
6
Make a 12.5% SDS PAGE gel using low fluorescence glass plates. The gel
should be made with wells into which the samples will be loaded.
7
Load each protein serial dilution in successive lanes on the gel.
8
Run the samples until the Bromophenol Blue dye front has nearly reached
the bottom of the gel.
9
Thoroughly clean the outside of the glass plates with double distilled
water.
10 Scan the gel at the appropriate wavelength with Typhoon Variable Mode
Imager or Ettan DIGE Imager.
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91
A Testing cell lysates for successful labeling
A.1 Testing new cell lysate for successful labeling
11 Insert the gel into the scanner in the correct orientation, see Chapter 7,
Image acquisition.
Fig A-1. CyDye DIGE Fluor Cy5 minimal dye scanned image
12 Quantify the labeling of each protein sample using ImageQuant TL
software.
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Testing cell lysates for successful labeling A
13 Carry out the statistics by opening the image in ImageQuant TL software.
Draw a single box over the first lane using the Object:Rectangle Tool.
Copy and Paste the rectangle for all of the samples that need to be tested
in the remaining lanes.
Fig A-2. CyDye DIGE Fluor Cy5 minimal dye scanned image. Lanes are
overlaid with identical boxes to give a volume report in ImageQuant TL Toolbox.
Ettan DIGE System User Manual 18-1173-17 Edition AB
93
A Testing cell lysates for successful labeling
A.1 Testing new cell lysate for successful labeling
14 In Analysis:Volume Report Setup highlight the boxes Object Name,
Volume, Area and select Results Only in the Print Format section.
15 Generate a volume report by clicking Analysis:Volume Report... in the
drop down menu.
16 Select all the relevant RECT in the Inspector window so that they are
highlighted blue.
17 Determine the labeling efficiency by comparing the volume of the new
protein samples and the control sample, which are on the same gel. The
labeling efficiency of these should be equivalent.
If labeling is comparable between the control and the new protein lysates
tested then samples can now be run on 2D gels. See the decision tree in
Fig A-3.
To investigate the cause of the problem, post-stain the gel with Deep
Purple Protein Total Stain. See Section 9.5, for information.
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Testing cell lysates for successful labeling A
Protein
labeling
OK?
Yes
No
Go to 2D
pH OK?
Yes
No
Deep Purple stain
for loading
Re-pH, re-label
and run 1D
Equal loading?
Yes
Incompatible
reagent
No
Re-quantify
and re-run 1D
Test against
recommended
samples
Fig A-3. Decision tree for troubleshooting labeling using 1D gels.
Ettan DIGE System User Manual 18-1173-17 Edition AB
95
A Testing cell lysates for successful labeling
A.2 Recipes
A.2
Recipes
2× Gel loading buffer
Reagent
Quantity
Final concentration
Tris (1 M, pH 6.8)
12 ml
120 mM
Glycerol (87% [v/v])
23 ml
20% (v/v)
SDS (MW 288.38)
4g
4% (w/v)
DTT (MW 154.2)
3g
200 mM
Bromophenol Blue
A few grains
trace
Make up to 100 ml with distilled water
12.5% 1D SDS PAGE gel composition
Reagent
Quantity for 100 ml of a 12.5% gel
Acrylamide/Bis 40% (w/v)
32.0 ml
Tris (1.5M, pH 8.8)
25.0 ml
10% (w/v) SDS
1.0 ml
10% (w/v) APS
1.0 ml
(undiluted) TEMED
*add immediately prior to use*
40 µl
Make up to 100 ml with distilled water
1× SDS electrophoresis running buffer
Reagent
Quantity
Final concentration
Tris (MW 121.14)
60.5 g
25 mM
Glycine (MW 75.07)
288 g
192 mM
SDS (MW 288.38)
20 g
0.1% (w/v)
Make up to 20 l with distilled water
Store at room temperature for up to 3 months.
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Labeling of cell surface proteins B
Appendix B
Labeling of cell surface proteins
B.1
Selective labeling of cell surface proteins
Cell surface proteins can be difficult to detect in a second dimension SDS PAGE
gel without fractionation or some other type of enrichment partly due to their low
abundance (1-2% of total cellular protein contents). They are often poorly
represented in second dimension SDS PAGE gels due to their hydrophobic nature
and high molecular weight.
The new protocol presented in this appendix for selective labeling of cell surface
proteins using CyDye DIGE Fluor minimal dyes makes it is possible to visually
enrich cell-surface proteins without performing sample fractionation. This
protocol is fast, simple to use and all three CyDye DIGE Fluor minimal dyes can be
used to label cell surface proteins.
These features allow for multiplexing using Ettan DIGE technology and analysis
of protein expression using DeCyder 2D software. In this way, the level of surface
proteins can be studied in different disease states or when responding to different
treatments. Small changes in abundance can be detected with high accuracy,
and results are supported by defined statistical methods.
Protocol
1
Grow the cells of interest until a minimal cell number of 5-10 x 106 cells
confluent or cells in suspension) is reached. For cells growing in
suspension, proceed to step 3.
2
Carefully detach adherent cells non-enzymatically (cell dissociation
media, enzyme free PVS-based).
3
Count the cells and pellet them by centrifugation. From now on, keep the
cells on ice.
4
Wash the cells by resuspending the pellet in 1 ml cold HBSS pH 8.5 buffer
and transfer to 1.5 ml eppendorff tube. Centrifuge the suspension at 800
x g and +4°C for 2 minutes.
5
Remove the supernatant and resuspend a cell pellet containing 5-10 x
106 cells in 200 µl cold labeling buffer (HBSS pH 8.5, 1M Urea). Add 1.5 µl
CyDye working solution (600 pmoles). Mix briefly by vortexing at slow
speed.
6
Label the cell surface for 20 minutes on ice in darkness. Then add 20 µl of
20 mM lysine and incubate another 10 minutes.
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97
B Labeling of cell surface proteins
B.1 Selective labeling of cell surface proteins
7
After labeling and quenching, wash the cells twice in 500 µl cold HBSS pH
7.4 by centrifugation at 800 x g and +4 °C for two minutes.
8
Resuspend the pellets directly in 150 µl cold lysis buffer (7M Urea, 2M
Thiourea, 4% CHAPS, 30 mM Tris, 5 mM MgAc pH 8.5) and leave on ice for
at least 1 hour with occasional vortexing. Centrifuge the lysate at
10 000 x g and +4°C for 5 minutes. Transfer the supernatant to a new
tube.
Note: Fractionation of the sample may be desired. It is not necessary for
improved detection of cell surface proteins but could be used to
verify lack of labeling of cytosolic proteins. Then, resuspend the cell
pellet in 150 µl cell lysis buffer included in the membrane
fractionation kit (2D Sample Prep for Membrane Proteins, Pierce).
After fractionation the sample can be directly applied on 2D
electrophoresis.
9
98
Add 2X Sample buffer to the lysed cell surface labeled sample. Now the
sample is ready for 2D electrophoresis. Proceed with the recommended
protocols for 2D DIGE electrophoresis.
Ettan DIGE System User Manual 18-1173-17 Edition AB
Reagents tested for compatibility with Ettan DIGE system C
Appendix C
Reagents tested for compatibility with Ettan
DIGE system
This section contains examples of reagents commonly used in 2D electrophoresis
experiments which have been tested for their compatibility with labeling using
CyDye DIGE Fluor dyes. They all have the DIGE approved seal attached.
This is not a complete list of reagents; if unlisted reagents or a combination of
these reagents are required in the cell lysis buffer it is recommended that the
labeling efficiency is checked following the instructions in Appendix A. These
examples are only intended as a guide.
C.1
List of reagents
Reducing agents
DL-dithiothreitol (DTT)
2 mg/ml - slight reduction in labeling
5 mg/ml - 2× reduction in labeling
10 mg/ml - 10× reduction in labeling
CyDye DIGE Fluor minimal dyes will react with
thiols at high concentration.
Tris-(2-carboxyethyl)
phosphine (TCEP)
0.5 to 1 mM - slight reduction in labeling
ß-mercaptoethanol
Significantly reduces labeling
2 mM - significant reduction in labeling
Detergents
Triton™ X-100
use at 1%
17% reduction in
labeling
NP40
up to 1%
No effect on labeling
SDS
up to 1%
No effect on labeling
Salts
Application of sample during
rehydration
<10 mM recommended
Application of sample via cup-loading
<50 mM recommended
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C Reagents tested for compatibility with Ettan DIGE system
C.1 List of reagents
Buffers
Tris
Recommend 10-40 mM, pH 8.0 - 9.0
pH is very important. pH 8.5 is optimal.
HEPES
Can cause focusing problems therefore not
recommended.
Bicarbonate
5 mM, pH 8.5 is acceptable
CHES
5 mM, pH 9–9.5 is acceptable
PBS
150 mM phosphate buffered saline
Tris sucrose
250 mM sucrose, 10 mM Tris
Protease Inhibitors
For all protease inhibitors: Mix compatible proteases at recommended
concentrations.
100
4-(2-aminoethyl)
benzenesulphonyl fluoride
(AEBSF) (Pefabloc™)
Causes charge trains unless protector reagent
is used
Complete™ protease inhibitor
cocktail
This product contains AEBSF, so the same
restrictions as above apply
Aprotinin
Compatible at recommended concentrations
(4-amidino-phenyl) methane
sulphonyl fluoride (APMSF)
Compatible at manufacturer's recommended
concentrations
EDTA
Compatible between 0.5-10 mM
Phenylmethylsulphonyl
fluoride (PMSF)
Compatible at manufacturer's recommended
concentrations
Pepstatin A
Compatible at manufacturer's recommended
concentrations
Protease inhibitor mix
Compatible at recommended concentrations
Ettan DIGE System User Manual 18-1173-17 Edition AB
Reagents tested for compatibility with Ettan DIGE system C
Phosphatase inhibitors
Phosphatase inhibitor
cocktail 1 (Sigma)
Compatible at manufacturer's recommended
concentrations
Phosphatase inhibitor
cocktail 2 (Sigma)
Compatible at manufacturer's recommended
concentrations
Sample preparation kits
2D Clean up kit
Compatible at manufacturer's recommended
concentrations
SDS PAGE Clean up kit
Compatible at manufacturer's recommended
concentrations
2D Quant kit
Compatible at manufacturer's recommended
concentrations
Sample grinding kit
Compatible at manufacturer's recommended
concentrations
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C Reagents tested for compatibility with Ettan DIGE system
C.1 List of reagents
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Trouble shooting guide D
Appendix D
Trouble shooting guide
The aim of this Appendix is to provide a help guide for problems that might be
encountered when running experiments. For general 2D troubleshooting
problems please refer to 2D Electrophoresis Principles and Methods handbook.
D.1 Sample preparation and labeling
Problem
Cause
Remedy
Low protein
yields from
the cell
lysate
Lysis procedure
Ensure cell culture densities were
optimal for cell lysis using sonication
Ensure cell pellet was not lost after
centrifugation
Ensure sonication was carried out for
long enough
Insufficient protein in
sample (protein concentration <1 mg/ml)
Remake protein lysate or concentrate
sample by precipitation with Ettan 2D
Clean-Up Kit (code no. 80-6484-51)
Horizontal
streaking
Low pH prior to
labeling
Check that pH is within range 8-9
immediately prior to labeling
Unexpected
protein
spots
present in
the gel
Contaminant proteins
have been introduced
into the sample prior
to the labeling
reaction
Check that gloves are used throughout
the procedure
Protein
spots
detected
more
strongly
with one
dye
Proteins not
denatured or
solubilized sufficiently
Use combination of chaotrope in lysis
buffer, such as 7 M urea/2 M thiourea
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D Trouble shooting guide
D.1 Sample preparation and labeling
104
Problem
Cause
Remedy
Weak
fluorescent
signal on 2D
gel image
Insufficient sample
buffering
Use 30 mM Tris to give sufficient
buffering capacity
Thiol agents present
in the sample
competing for dye.
Check if
concentration of DTT
is >2 mg/ml in protein
sample preparation
method
Dilute protein lysate with DTT-free lysis
buffer. Clean sample by precipitation
with Ettan 2D Clean-Up Kit, or increase
the amount of dye in the labeling
reaction
Primary amines such
as Pharmalytes or
ampholytes are
present in sample
during labeling,
competing for CyDye
DIGE Fluor minimal
dye
Dilute protein lysate with amine-free
lysis buffer. Clean sample by
precipitation with Ettan 2D Clean-Up Kit,
or increase the amount of dye in the
labeling reaction
Incorrect
concentration of
protein in lysate
Use a detergent or thiourea compatible
protein assay kit, e.g. Protein
Determination Reagent or Ettan 2D
Quant Kit
Check quality of DMF
Should be >99.8% anhydrous DMF from
a bottle that has not been open for
longer than 3 months
Degraded dye due to
hydrolysis of NHSester or
photodegradation of
fluorophore
Ensure that the appropriate storage
conditions have been used for the
CyDye DIGE Fluor minimal dye (in dark
at –20°C). Check specific batch expiry
date and reconstitution date
Incorrect dye:protein
ratio used
400 pmol of dye per 50 µg of protein is
recommended. If there is a large
concentration of other components
which can react with the dye, then more
dye (up to 2 nmol per 50 µg of protein)
can be used
Low pH prior to
labeling
Check pH is 8.5 immediately prior to
labeling. If necessary, increase pH using
higher pH lysis buffer containing 30 mM
Tris (pH 9.0-10.0) or use 50 mM NaOH
Ettan DIGE System User Manual 18-1173-17 Edition AB
Trouble shooting guide D
D.2 First dimension electrophoresis
Problem
Cause
Remedy
Current is
zero or too
low
External electrode
contacts are poor
Ensure that the electrodes at the
bottom of the strip holder (one at each
end) make metal-to-metal contact with
the appropriate electrode area
Internal electrode
contacts are poor
Ensure that the gel makes contact with
both electrodes in the strip holder
Immobiline DryStrips
not fully rehydrated
Check that the Immobiline DryStrips are
fully rehydrated along their entire
length. Electrical contact at the
electrodes is reduced by incomplete
rehydration
No conduction
through electrode
wicks
Check that the electrode wicks (if used)
were moistened prior to use
Voltage too
low or does
not reach
the
maximum
set value
Current limit setting is
incorrect
Check that the current limit is properly
set
Incorrect number of
Immobiline DryStrips
Check that the correct number of strips
in place is set on the IPGphor program
No Pharmalytes
added to sample
The recommended Pharmalyte
concentration is 1.0% (v/v)
Sparking or
burning in
strips
High current
Do not exceed the recommended
setting of 50 µA per Immobiline DryStrip
Strips not rehydrated
Check that Immobiline DryStrips are
fully rehydrated along their entire
length
Presence of bubbles
Check that any large bubbles trapped
under the Immobiline DryStrips after
wetting with rehydration solution are
removed prior to focusing
Strips drying out
during focusing
Ensure that sufficient Immobiline
DryStrips Cover Fluid has been applied
High salt
concentration
Clean sample to remove excess salts
Poor electrical
contact in first
dimension
Check that the strips are in contact with
the strip holder electrodes
Proteins
have not
focused
Ettan DIGE System User Manual 18-1173-17 Edition AB
105
D Trouble shooting guide
D.3 Second dimension electrophoresis
D.3 Second dimension electrophoresis
106
Problem
Cause
Remedy
No protein
spots are
visible on
the gel
Incorrect labeling
protocol
Check the sample preparation and
labeling protocol
Inefficient sample
solubilization
Increase concentration of solubilizing
components in the sample solution. The
upper concentration limits for common
reagents are: Urea 9.8 M, Thiourea 2 M,
zwitterionic detergent CHAPS 4%,
Pharmalyte 1%, DTT 2-3%
No or insufficient SDS
in electrophoresis
running buffer
Ensure electrophoresis running buffer is
correctly formulated
Individual
spots
appear as
multiple
bands or
are missing,
unclear, or
in the wrong
position
Immobiline DryStrips
placement
Ensure that the plastic backing of the
Immobiline DryStrips are against the
glass plate on the second dimension gel,
directly onto the top of the acrylamide
gel
Protein oxidation
during
electrophoresis
Prevent oxidation of oxygen-sensitive
proteins in the gel. Check the correct
equilibration conditions are used prior
to the second dimension separation.
DTT reduction, then treatment with
iodoacetamide alkylates the sulphydryl
groups and thus prevents the reduced
proteins from re-oxidizing
Formation
of charge
trains
Protein
carbamylation
Check that all solutions containing urea
were prepared freshly and ensure that
all solutions containing urea were not
heated above 37° C at any time
Horizontal
streaking or
incompletely focused
spots
High sample load
Reduce sample load by adding less
sample to the rehydration solution
Insufficient focusing
time
Increase total Vh for focusing
Ettan DIGE System User Manual 18-1173-17 Edition AB
Trouble shooting guide D
Problem
Cause
Remedy
Vertical
streaking or
incompletely focused
spots
SDS depletion during
second dimension
electrophoresis
Use 0.2% SDS in the running buffer for
both top and bottom buffer tanks.
Over-alkylated
proteins
Use lower pH, higher DTT concentration
or lower iodoacetamide concentration
when equilibrating Immobiline DryStrips
Sample
insolubility
and
particulates
Insufficient chaotrope
or detergent
Ensure the correct solubilization solution
has been used
Cloudy samples
Remove insoluble material from the
sample using ultracentrifugation
Poor first
dimension
focusing
Ionic detergent
concentration too
high in lysis buffer
If SDS is used in sample preparation, the
final concentration must not exceed
0.25% after dilution into the rehydration
solution. Also ensure that the nonionic
detergent is present in a concentration
at least 8 times higher than the
concentration of any ionic detergent to
ensure complete removal of SDS from
the proteins
Streaking or
smearing
Sample rich in nucleic
acids
Add DNase and RNase, or sonicate to
hydrolyze nucleic acids
Sample
aggregation
or precipitation
Focusing conditions
not optimized
Program a low initial voltage that
increases gradually, and/or increase
time at maximum voltage.
Ettan DIGE System User Manual 18-1173-17 Edition AB
Ensure that the gel has been prepared
with the correct concentration of SDS
Extended focusing may result in electroendosmosis where water and protein
movement can produce horizontal
streaking. Minimize water transport by
employing a maximum
pH range Immobiline DryStrips and
apply electrode pads
107
D Trouble shooting guide
D.4 Typhoon Variable Mode Imager results
D.4 Typhoon Variable Mode Imager results
For a complete guide to troubleshooting Typhoon Imager results, please refer to
Typhoon User Guide.
108
Problem
Cause
Remedy
Protein
spots do not
show up on
the gel
image
The wrong laser and
emission filters have
been selected for the
CyDye DIGE Fluor
minimal dye used
Select correct laser and filter for each
CyDye DIGE Fluor minimal dye
The labeling reaction
has not been
performed correctly
Reconstitute stock dye or make fresh
working dye solution in fresh DMF.
Repeat the labeling
The PMT voltage is
too low
Rescan with higher PMT voltage
The gel
image
appears
black
The PMT voltage is
too high
Rescan with lower PMT voltage
Appearance
of
nonspecific
background
on gel
image
Platen contaminated
with dye from
scanning other gels
Clean platen. Rescan gel on different
part of platen to confirm that
background does not move with the gel
Bacterial/
mycoplasma
contamination in gel
running equipment
Bacterial/mycoplasma contamination
in gel running equipment
Appearance
of small
spots (sharp
peaks in gel
image)
Dirt and dust on gel
plates or platen
Clean platen or gel plates. Rescan gel on
different part of platen to confirm that
background does not move with gel
Dirt in gel
Filter acrylamide
DeCyder 2D
software
gives poor
spot
boundaries
Incorrect scan
resolution
Rescan gels with resolution set to
100 µm
Faint, illdefined gel
images,
possibly
with high
background
Incorrect focal plane
selected
Re-scan with correct focal plane
selected. Setting gel orientation and
scan resolution
Ettan DIGE System User Manual 18-1173-17 Edition AB
Index
Index
A
Analytical workflow ............................................................................................................................................. 10
B
Batch Processor ..................................................................................................................................................... 76
BVA ............................................................................................................................................................................... 76
C
Cleaning glass plates .......................................................................................................................................... 81
Cropping .................................................................................................................................................................... 71
CyDye DIGE Fluor dyes
reconstitution .............................................................................................................................................. 34
stability of stock dye solution .............................................................................................................. 34
when to use which dye ........................................................................................................................... 14
working solution ......................................................................................................................................... 35
CyDye DIGE Fluor minimal dyes
description ........................................................................................................................................... 16, 18
labeling reaction ........................................................................................................................................ 16
stock solution dilution .............................................................................................................................. 35
CyDye DIGE Fluor saturation dyes
labeling reaction ........................................................................................................................................ 17
D
DeCyder 2D software .......................................................................................................................................... 75
Deep Purple
post-staining ....................................................................................................................................... 66, 84
DIA ................................................................................................................................................................................ 76
DIGE File Naming Format .................................................................................................................................. 68
Displacing solution ...................................................................................................................................... 52, 58
E
EDA .............................................................................................................................................................................. 76
Equilibration of focused Immobiline DryStrips .............................................................................. 52, 54
Equilibration solution 1 ....................................................................................................................................... 59
Equilibration solution 2 .............................................................................................................................. 54, 60
Ettan DALT ................................................................................................................................................................ 51
Ettan DIGE Imager ................................................................................................................................................ 73
Ettan IPGphor 3 Isoelectric Focusing System .......................................................................................... 41
Experimental design
inter-gel matching .................................................................................................................................... 20
internal standard .............................................................................................................................. 18, 27
I
IEF
preparativ workflow ................................................................................................................................. 81
rehydration of Immobiline DryStrips ................................................................................................ 43
running on Ettan IPGphor 3 instrument .......................................................................................... 47
running preparations ............................................................................................................................... 45
sample application protocol selection ............................................................................................ 43
workflow ........................................................................................................................................................ 42
ImageMaster 2D Platinum software ............................................................................................................ 77
Ettan DIGE System User Manual 18-1173-17 Edition AB
109
Index
ImageQuant TL
cropping .........................................................................................................................................................71
trouble shooting .........................................................................................................................................72
Immobiline DryStrip
equilibration .........................................................................................................................................52, 54
rehydration ...................................................................................................................................................43
Reswelling Tray ..................................................................................................................................43, 44
L
Labeling
CyDye DIGE Fluor minimal dyes ................................................................................................16, 36
CyDye DIGE Fluor saturation dyes ...........................................................................................17, 37
testing sample labeling ..................................................................................................................33, 91
workflow .........................................................................................................................................................30
M
Manuals for related products ..........................................................................................................................12
P
PMT ......................................................................................................................................................................67, 70
Post-staining ............................................................................................................................................................84
Preparative gel .......................................................................................................................................................81
Preparative gel staining .....................................................................................................................................79
Preparative workflow ..........................................................................................................................................80
preparing the labeled protein samples for the first dimension .................................................... 110
R
Reference markers ...............................................................................................................................................83
Result variation .......................................................................................................................................................13
S
Sample preparation
cell wash solution .......................................................................................................................................30
preparative workflow ...............................................................................................................................81
preparing the labeled protein samples for the first dimension ............................................30
protein quantitation ..................................................................................................................................31
requirements for lysis buffer ................................................................................................................30
Scanning
emission filters .............................................................................................................................................66
file output ..............................................................................................................................................62, 70
fluorescence acquisition mode ...........................................................................................................65
fluorescence scan parameters ............................................................................................................65
gel alignment guide ..................................................................................................................................63
gel orientation .............................................................................................................................................67
image cropping ...........................................................................................................................................71
monitoring scan progress ......................................................................................................................70
pixel size .........................................................................................................................................................67
PMT voltage ..................................................................................................................................................66
press sample ................................................................................................................................................67
select scan area ..........................................................................................................................................65
sensitivity .......................................................................................................................................................66
SDS electrophoresis running buffer ..............................................................................................................59
SDS Equilibration buffer-stock solution .............................................................................................54, 59
Second dimension SDS PAGE
casting gels ...................................................................................................................................................52
110
Ettan DIGE System User Manual 18-1173-17 Edition AB
Index
loading of focused Immobiline DryStrips .............................................................................. 52, 55
low fluorescence glass plates .............................................................................................................. 52
preparative workflow .............................................................................................................................. 81
running buffer ............................................................................................................................................. 55
storage of gels post electrophoresis ................................................................................................ 57
storage of gels prior to separation .................................................................................................... 53
Standard cell lysis solution ............................................................................................................................... 30
T
Trouble shooting .................................................................................................................................................103
Typhoon Variable Mode Imager .................................................................................................................... 61
cleaning .......................................................................................................................................................... 62
instrument ..................................................................................................................................................... 61
placing gels .................................................................................................................................................. 62
scan parameters ........................................................................................................................................ 64
scanning ........................................................................................................................................................ 68
workflow ........................................................................................................................................................ 62
X
XML Toolbox ............................................................................................................................................................ 76
Ettan DIGE System User Manual 18-1173-17 Edition AB
111
Index
112
Ettan DIGE System User Manual 18-1173-17 Edition AB
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User Manual 18-1173-17 AB
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