2-D Sample Prep + 2-D Electrophoresis + Imaging

2-D Sample Prep + 2-D Electrophoresis + Imaging
Sample Prep + 2-D Electrophoresis + Imaging
2-D
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2-D Electrophoresis Workflow
How-To Guide
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Positioning Guide
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2-D Electrophoresis Guide
Table of Contents
Part I: Theory and Product Selection
Chapter 1 Overview of
Two-Dimensional Electrophoresis
5
The Context of Proteomics
6
Overview of Experimental Design
6
8
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9
Sample Preparation
First-Dimension Separation: IEF
Second-Dimension Separation: SDS-PAGE
Detection
Image Acquisition, Analysis, and Spot Cutting
Protein Digestion and Identification by Mass Spectrometry
Chapter 2 Sample Preparation
11
The Importance of Sample Preparation
12
General Considerations
12
Cell Lysis
12
Protein Solubilization
15
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17
Chaotropic Agents
Detergents
Reducing Agents
Ampholytes, Buffers, and Other Additives
Removal of Interfering Substances
General Considerations
Nucleic acids (DNA and RNA)
Polysaccharides
Phenolic Compounds
Lipids
Salts and Other Small Ionic Compounds
Prevention of Keratin Contamination
Prefractionation
About This Guide
This guide describes the experimental methods and tools used in 2-D
electrophoresis and proteomics research. It provides background information
about technologies common to all proteomics studies as well as protocols and
advice you can use as a starting point for your studies. This guide also explains
how experimental conditions can be varied and interpreted to optimize your
results and provides an extensive set of references that you can consult for
more information. Since each sample, experimental approach, and objective
is different, this guide offers ideas for developing customized protocols suitable
for the analysis of your samples.
5
Fractionation by Subcellular Location
Fractionation by Solubility/Hydrophobicity
Fractionation by Protein Charge
Fractionation by pI
Fractionation by Size (MW)
Depletion and Dynamic Range Reduction
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Depletion
Dynamic Range Reduction
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ProteoMiner Technology
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Sample Quantitation (Protein Assays)
30
Chapter 3 The First Dimension:
Isoelectric Focusing (IEF)
33
Protein Separation by Isoelectric Point (pI)
34
IEF Media: IPG Strips vs. Carrier Ampholytes
35
Selection of IPG Strips
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Choice of pH Gradient
Choice of IPG Strip Length
Estimation of pl
37
Sample Application
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Sample Application during Rehydration
Sample Application by Cup Loading
Setup for IEF
39
Power Conditions for IEF
40
Chapter 4 The Second Dimension:
SDS-PAGE
43
Protein Separation by Size
44
Selection of Polyacrylamide Gels
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Choice of Gel Percentage (Composition)
Choice of Gel Size
Choice of Buffer System
Transition from First to Second Dimension
52
Power Conditions and Reagents for SDS-PAGE
52
Molecular Weight Estimation
53
Chapter 5 Detection
55
Detection of Proteins in Gels
Coomassie Stains
Silver Stains
Fluorescent Stains
Negative Stains
Stain-Free Technology
Detection of Proteins on Western Blots
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Bio-Rad’s Proteomics Program
From sample preparation to protein analysis, Bio-Rad’s tools provide
you with choices in methodology, protocols, and products. Our informative
2-D Electrophoresis and Analysis Applications and Technologies web pages
are a valuable resource with video tutorials, protocols, troubleshooting tips,
and much more. To learn more, visit www.bio-rad.com/2DElectroAnalysis.
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2-D Electrophoresis Guide
Table of Contents
Chapter 6 Image Acquisition, Analysis,
and Spot Cutting
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85
Isoelectric Focusing
112
Tips for IEF
86
SDS-PAGE
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IPG Strip Rehydration and Sample Loading
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Total Protein Staining
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Performing IEF
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2-D Gel Evaluation
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Part IV: Appendices
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Finding Protein Spots of Interest
64
Image Acquisition
Image Analysis
Image Optimization, Spot Detection, and Quantitation
Gel Comparison
Data Normalization
Data Analysis and Reporting
Spot Cutting from 2-D Gels
67
Chapter 7 Identification and
Characterization of 2-D Protein Spots
69
Beyond Excision
70
Proteolytic Digestion
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Washing
Reduction and Alkylation
In-Gel Proteolytic Digestion
Identification by Mass Spectrometry
Peptide Mass Fingerprinting
Tandem Mass Spectrometry (MS/MS)
Establishment of 2-D Databases
Part II: Methods
Chapter 8 Sample Preparation
Tips for Sample Preparation
Lysis (Cell Disruption)
Protein Solubilization
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Cell Lysis and Protein Extraction Procedures
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Suspension Cultured Human Cells
Monolayer Cultured Human Cells
Mammalian Tissue
Microbial Cultures
Plant Leaves
Sample Cleanup
Buffer Exchange (Desalting)
Sample Quantitation (RC DC Protein Assay) Microfuge Tube Assay Protocol (1.5 ml)
IPG Strip Rehydration in Rehydration/Equilibration Trays
Followed by IEF
IEF with Gel-Side Up
IEF with Gel-Side Down
Cup Loading (IEF with Gel-Side Up)
IPG Strip Rehydration in the Focusing Tray Followed by IEF
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Appendix A
Glossary
90
Appendix B
Chapter 10 Second-Dimension SDS-PAGE
95
References
Related Bio-Rad Literature
Tips for SDS-PAGE
96
Appendix C
IPG Strip Equilibration
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Sealing IPG Strips onto SDS-PAGE Gels
98
SDS-PAGE
99
IEF Programming Recommendations
Buffers and Solutions
2
Part III: Troubleshooting
Chapter 9 First-Dimension IEF
with IPG Strips
Chapter 11 Protein Detection
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Tips for Total Protein Staining
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Long-Term Storage of Stained Gels
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Total Protein Staining
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Bio-Safe Coomassie Stain
Flamingo Fluorescent Gel Stain
Oriole Fluorescent Gel Stain
SYPRO Ruby Protein Gel Stain
Silver Stain Plus Kit
Chapter 12 In-Gel Trypsin Digestion
Tryptic Digestion Protocol
Reagents and Solutions
Destaining Gel Plugs from Silver-Stained Gels (Pre-Treatment)
General Destaining Protocol Reduction and Alkylation Protocol
Digestion Protocol
Extraction Protocol
Ordering Information
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2-D Electrophoresis Guide
Theory and Product Selection
PART I
Theory and
Product Selection
CHAPTER 1
Overview of
Two-Dimensional
Electrophoresis
4
5
Chapter 1: Overview of Two-Dimensional Electrophoresis
2-D Electrophoresis Guide
The Context of Proteomics
Proteome analysis (proteomics) is the comprehensive
analysis of proteins present in a sample and
representing a particular physiological state at a
particular point in time. The aim of proteomics is to
determine the presence, relative abundance, and
posttranslational modification state of a large fraction
of the proteins in a sample (Wilkins et al. 1996).
Since proteins are directly involved in cellular structure,
regulation, and metabolism, proteomics can often yield
a more informative and accurate picture of the state of
a living cell than can analysis of the genome or mRNA.
One of the greatest challenges of proteome analysis
is the reproducible separation of complex protein
mixtures while retaining both qualitative and
quantitative relationships. Many combinations of
techniques can be used to separate and analyze
proteins, but two-dimensional (2-D) electrophoresis
is uniquely powerful in its ability to separate hundreds
to thousands of products simultaneously (Choe and
Lee 2000). This technique uses two different
electrophoretic separations, isoelectric focusing (IEF)
and SDS-PAGE, to separate proteins according to
their isoelectric point (pI) and molecular weight.
The identities of individual protein spots from the gel
can then be identified by mass spectrometry (MS)
of their tryptic peptides. Together with computerassisted image evaluation systems for comprehensive
qualitative and quantitative examination of proteomes,
proteome analysis also allows cataloguing and
comparison of data among groups of researchers.
Other common methods of proteome analysis involve
the proteolytic digestion of sample proteins and the
chromatographic separation of the resulting peptides
coupled directly to mass spectrometric analysis.
Peptides are identified by referencing a database,
and their proteins of origin are inferred. While these
methods are largely automatable and provide an
impressive depth of proteome coverage, some
information is lost when analyzing protein fragments
instead of intact proteins. The 2-D electrophoresis
approach maintains proteins in their intact states and
enables the study of isoform distribution, which is not
possible if the sample is proteolytically digested prior
to separation. Since proteins can be selected through
image analysis, mass spectrometry need be applied
only to the proteins of interest. This is an important
consideration when access to instrumentation or the
expense of mass spectrometric analysis is a limitation.
The suitability of 2-D electrophoresis to proteome
analysis is clear, but its applications also extend to
biomarker detection, development of drug and other
therapies, and optimization and development of
protein purification strategies.
Overview of Experimental Design
The general workflow in a 2-D electrophoresis
experiment (Figure 1.1) and some of the factors
affecting the way the experiment is performed
are outlined next.
Theory and Product Selection
2-D Electrophoresis Workflow
Sample Preparation
Prepare the protein at a concentration and in a solution suitable
for IEF. Choose a method that maintains the native charge,
solubility, and relative abundance of proteins of interest.
First-Dimension Separation: IEF
Separate proteins according to pI by IEF. Select the appropriate IPG
strip length and pH gradient for the desired resolution and sample
load. Select appropriate sample loading and separation conditions.
Second-Dimension Separation: SDS-PAGE
Separate proteins according to size by SDS-PAGE.
Select the appropriate gel size and composition
and separation conditions.
Detection
Visualize proteins using either a total protein stain or fluorescent
protein tags. Select a staining technique that matches
sensitivity requirements and available imaging equipment.
Image Acquisition and Analysis
Capture digital images of the 2-D protein patterns
using appropriate imaging equipment and software.
Then analyze the patterns using 2-D analysis software.
Protein Excision, Digestion, and Identification
Excise protein spots of interest from the gel,
digest the proteins, and analyze the digests by MS.
Fig. 1.1. General workflow for a 2-D experiment.
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Chapter 1: Overview of Two-Dimensional Electrophoresis
2-D Electrophoresis Guide
Sample Preparation
Effective sample preparation is key for the success
of the experiment. The sample dictates the type of
extraction technique used, and the solubility, charge,
and pI of the proteins of interest affect the method
of solubilization. The protein fraction used for 2-D
electrophoresis must be solubilized in a denaturing
solution of low ionic strength; this solution cannot
contain components that alter protein size or charge.
Sample preparation also involves optional steps to
deplete abundant proteins, reduce the complexity
of the protein mixture, or select a subproteome of
interest. Details and recommendations for sample
preparation can be found in Chapter 2.
First-Dimension Separation: IEF
In 2-D electrophoresis, the first-dimension separation
step is IEF. Proteins are separated electrophoretically
on the basis of their pI, the pH at which a protein
carries no net charge. For general proteome analysis,
IEF is best performed in an immobilized pH gradient
(IPG) strip and under conditions aimed at completely
denaturing and solubilizing all the proteins in the
sample (as opposed to native IEF, which aims to
preserve native structures and activities). Chapter 3
discusses IEF.
Second-Dimension Separation: SDS-PAGE
The second-dimension separation step is SDS-PAGE,
where the proteins already separated by IEF are further
separated by their size. Prior to second-dimension
separation, an equilibration step is applied to
the IPG strip containing the separated proteins.
This process reduces any disulfide bonds that may
have re-formed during the first dimension and alkylates
the resultant sulfhydryl groups. Concurrently, the
proteins are complexed with SDS for separation on
the basis of size. Following electrophoretic separation
on a slab gel, the result is a two-dimensional array
of separated protein “spots” (Figure 1.2). Seconddimension SDS-PAGE is discussed in Chapter 4.
Theory and Product Selection
Detection
Proteins separated in gels are usually not visible to
the naked eye and must, therefore, be either stained
or labeled for visualization. Several factors determine
the best choice of staining method, including desired
sensitivity, linear range, ease of use, expense, and the
type of imaging equipment available. There is no ideal
universal stain. Sometimes proteins are detected after
transfer to a membrane support by western blotting.
These topics are discussed in Chapters 5 and 6.
Image Acquisition, Analysis, and Spot Cutting
The ability to collect data in digital form is one of the
major factors that make 2-D gels a practical means
of collecting proteome information. It allows the
unbiased comparison of samples and gels, transfer of
information among research groups, and cataloguing
of data. Many types of imaging devices interface with
software designed specifically to collect, interpret,
and compare proteomics data.
Once interesting proteins are selected by differential
analysis or other criteria, the proteins can be excised
from gels and identified by mass spectrometry.
The ExQuest™ spot cutter, which can be operated
independently or programmed to run from PDQuest™
software, automatically cuts selected protein spots
from gels with precision and deposits them into the
wells of microplates.
Sample Preparation
Imaging equipment, software, and the ExQuest spot
cutter are discussed in Chapter 6.
Protein Digestion and Identification by
Mass Spectrometry
First Dimension
Isoelectric focusing (IEF), separation by pl
Low pH
Second Dimension
SDS-PAGE,
separation by MW
High pH
High MW
The excised gel plugs are destained and enzymatically
digested (usually with trypsin) in preparation for
identification by mass spectrometry. The use of mass
spectrometry for precise mass and partial sequence
determination, coupled with the availability of protein
sequence databases, has made high-throughput
protein identification possible. An overview of this
process is provided in Chapter 7.
Low MW
Fig. 1.2. 2-D electrophoresis. Protein spots result from two separations: first by pI (IEF) and then by size (SDS-PAGE).
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2-D Electrophoresis Guide
Theory and Product Selection
CHAPTER 2
Sample Preparation
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Chapter 2: Sample Preparation
2-D Electrophoresis Guide
The Importance of Sample Preparation
Sample preparation contributes significantly to
the overall reproducibility and accuracy of protein
expression analysis (Link 1999, Rabilloud 1999,
Molloy 2000). Without proper sample preparation,
proteins may not separate from one another or may
not be represented in the 2-D pattern.
A successful sample preparation strategy enhances
separation quality by:
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ffectively and reproducibly solubilizing proteins
E
of interest
reventing protein aggregation and loss of solubility
P
during IEF
reventing proteolysis or other chemical or
P
enzymatic protein modifications
emoving or minimizing the effect of contaminants
R
such as salts, detergents, nucleic acids, and other
interfering molecules
ielding proteins of interest at detectable levels,
Y
which may require fractionation to reduce protein
sample complexity or removal of interfering abundant
or irrelevant proteins
This chapter provides an overview of the principles and
recent developments in sample preparation strategies
prior to first-dimension IEF.
■■
■■
eep the sample preparation workflow as simple
K
as possible; increasing the number of sample
handling steps may increase variability and the
risk of sample loss
ith cell or tissue lysates, include protease inhibitors
W
to minimize artifacts generated by proteolysis;
protease inhibitors are generally not required for
samples like serum or plasma
Table 2.1. Suitability of cell disruption methods for various sample types.
etermine the amount of total protein in each
D
sample using a protein assay that is compatible
with chemicals in your samples
void freeze-thaw cycles; use protein extracts
A
immediately or aliquot them into appropriately sized
batches and store them at –70°C
Cell Lysis
The effectiveness of a cell lysis method determines
the accessibility of intracellular proteins for extraction
and solubilization. Different biological materials require
different lysis strategies, which can be divided into two
main categories: gentle methods and harsher methods
(Table 2.1).
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General Considerations
Since protein types and sample origins show great
diversity, there is no universal sample preparation
method. In addition, some proteins simply cannot
be solubilized under conditions compatible with IEF.
Sample preparation procedures must be optimized
empirically and tailored to each sample type and
experimental goal. The following general sample
preparation guidelines should be kept in mind:
olubilize proteins in a solution that is compatible
S
with IEF. Incubate proteins in 2-D lysis solution for
at least 30 min at room temperature (denaturation,
solubilization, and disaggregation are timedependent processes)
■■
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se gentle cell disruption protocols with cells that
U
lyse easily, such as blood cells and tissue culture cells
se harsher methods, which are based mainly on
U
mechanical rupture (Goldberg 2008), with biological
materials that have tough cell walls (for example,
plant cells and tissues, and some microbes)
hen working with a new sample, compare at least
W
two different cell disruption protocols with respect to
yield (by protein assay) and qualitative protein content
(by one-dimensional SDS-PAGE)
ptimize the power settings of mechanical rupture
O
systems and the incubation times of lysis approaches
echanical cell lysis usually generates heat;
M
use cooling where required to avoid overheating
the sample
A number of other components are often added to
disruption protocols. Sand, resin, or glass beads
facilitate the disruption of tissues and of plant and
yeast cell walls when added to manual grinding
procedures. Hypotonic buffers cause cells to burst
more readily under physical shearing, and enzymes
such as cellulase, pectinase, lyticase, and lysozyme
are added to break down plant, yeast, and bacterial
cell walls. Nucleases can be added to remove nucleic
acids, which can increase sample viscosity and
interfere with subsequent separation (see the Removal
of Interfering Substances section).
Yeast, Green
Mammalian
Algae, Plant
SoftCell
Technique
Description
Bacteria Fungi Seeds MaterialTissues Culture
Gentle Methods
Osmotic lysis
Suspension of cells in hypotonic solution;
cells swell and burst, releasing cellular contents
—
—
—
—
—
•
Freeze-thaw lysis
Freezing of cells in liquid nitrogen
and subsequent thawing
—
—
—
—
—
•
Detergent lysis
Suspension of cells in detergent-containing
solution to solubilize the cell membrane;
this method is usually followed by another
disruption method, such as sonication
—
—
—
—
—
•
Enzymatic lysis
Suspension of cells in iso-osmotic solutions containing enzymes that digest the cell wall
(for example, cellulase and pectinase for plant
cells, lyticase for yeast cells, and lysozyme for
bacterial cells); this method is usually followed by
another disruption method, such as sonication
•
•
—
•
—
—
Sonication
Disruption of a cell suspension, cooled on ice
to avoid heating and subjected to short bursts
of ultrasonic waves
•
•
—
—
—
•
French press
Application of shear forces by forcing a cell
suspension through a small orifice at high pressure
•
•
—
•
—
•
Grinding
Breaking cells of solid tissues and microorganisms
with a mortar and pestle; usually, the mortar is
chilled with liquid nitrogen and the tissue or cells
are ground to a fine powder
•
•
•
•
•
—
Mechanical
homogenization
Homogenization with either a handheld device
(for example, Dounce and Potter-Elvehjem
homogenizers), blenders, or other motorized
devices; this approach is best suited for soft,
solid tissues
—
—
—
•
•
—
Glass-bead
homogenization
Application of gentle abrasion by vortexing
cells with glass beads
•
•
—
—
—
•
Harsher Methods
All but the most gentle cell disruption methods destroy
the compartmentalization of a cell, causing the
release of hydrolases (phosphatases, glycosidases,
and proteases). These enzymes modify proteins in
the lysate, which complicates differential analysis.
The data generated by 2-D electrophoresis are only
meaningful when the integrity of the sample proteins
reflects the state in which they are found in the living
organism. Avoid enzymatic degradation by using one
or a combination of the following techniques:
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12
Theory and Product Selection
isrupt the sample or place freshly lysed samples in
D
solutions containing strong denaturing agents such
as 7–9 M urea, 2 M thiourea, or 2% SDS. In this
environment, enzymatic activity is often negligible
erform cell lysis at low temperatures to diminish
P
enzymatic activity
yse samples at pH >9 by adding a base such
L
as sodium carbonate or Tris(hydroxymethyl)aminomethane (Tris) to the lysis solution
(proteases are often least active at basic pH)
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dd protease inhibitors to the lysis solution.
A
Examples include either small molecules,
such as phenylmethylsulfonyl fluoride (PMSF),
aminoethyl-benzene sulphonyl fluoride (AEBSF),
tosyl lysine chloromethyl ketone (TLCK),
tosyl phenyl chloromethyl ketone (TPCK),
ethylenediaminetetraacetic acid (EDTA), and
benzamidine, or peptide protease inhibitors such
as leupeptin, pepstatin, aprotinin, and bestatin.
For best results, use a combination of inhibitors
in a protease inhibitor cocktail
If protein phosphorylation is to be studied, include
phosphatase inhibitors such as fluoride or vanadate
Following cell disruption:
■■
■■
heck the efficacy of cell disruption by light
C
microscopy (if the sample is a cell suspension)
entrifuge all extracts extensively (20,000 × g for
C
15 min at 15°C) to remove any insoluble material;
solid particles may block the pores of the IPG strip
13
Chapter 2: Sample Preparation
2-D Electrophoresis Guide
Protein Solubilization
Products for Cell Lysis and
Protein Extraction
ReadyPrep™ mini grinders contain a grinding
tube, grinding resin, and fitted pestle and offer an
easy, efficient mechanism for manually grinding
small biological samples. The grinding resin is a
neutral abrasive material made of a high-tensile
microparticle that does not bind protein or nucleic
acids. The combination of the pestle and resin
effectively disrupts animal or plant tissues and cells.
ReadyPrep mini grinders are available for purchase
separately or as components of the MicroRotofor™
cell lysis kits.
Proteins in a biological sample are often associated
with other proteins, integrated into membranes, or
parts of large complexes. Protein solubilization is the
process of breaking interactions involved in protein
aggregation (Rabilloud 1996), which include disulfide
and hydrogen bonds, van der Waals forces, and ionic
and hydrophobic interactions. If these interactions are
not disrupted, proteins can aggregate or precipitate,
resulting in artifacts or sample loss. For successful
2-D electrophoresis, proteins must be well solubilized.
ReadyPrep Mini Grinder
Bio-Rad also offers a range of kits for cell disruption
and protein extraction:
■■
■■
■■
icroRotofor lysis kits1 provide convenient,
M
effective methods optimized for the preparation
of protein samples from mammalian, plant, yeast,
and bacterial sources. Depending on the sample
type, these kits employ tissue maceration using
ReadyPrep mini grinders and/or solubilization into
a chaotropic extraction buffer
he ReadyPrep protein extraction kit (total protein)
T
uses the powerful zwitterionic detergent ASB-14
in a strongly chaotropic solubilization buffer to
prepare total cellular protein extracts suitable for
2-D electrophoresis (depending on sample type,
additional cell lysis protocols may be needed when
using this kit)
ther ReadyPrep protein extraction kits facilitate
O
extraction of specific classes of proteins and are
discussed later in this chapter
Such standardized lysis and extraction protocols
are often useful for initial proteomic analyses and
for consistent sample preparation.
1
14
MicroRotofor Lysis Kit
Sample lysis solutions typically contain a number
of compounds that meet the requirements, both
electrically and chemically, for compatibility with IEF.
To allow high voltages to be applied during IEF without
producing high currents, the compounds must not
increase the ionic strength of the solution. In some
cases, it may be necessary to prepare samples using
additives that facilitate protein solubilization but that
have limited compatibility with IEF (for example, salts
and SDS). In these cases, the potentially interfering
substance must be removed prior to sample
application, or actions must be taken to mitigate its
effect (see the Removal of Interfering Substances
section). See Chapter 9 for sample preparation
procedures and solutions; for a thorough discussion
of solubilization methods, refer to Rabilloud (2000).
Chaotropic Agents
ReadyPrep Protein Extraction Kit
Products for cell lysis and protein extraction. A number of other
ReadyPrep protein extraction kits facilitate disruption and extraction
of specific classes of proteins.
or added convenience, the extraction buffer included with these kits can also be used as the sample solution for IEF with the
F
MicroRotofor cell or with IPG strips.
These compounds disrupt hydrogen bonds and
hydrophobic interactions both between and within
proteins. When used at high concentrations,
chaotropic agents disrupt secondary protein
structure and bring into solution proteins that are
otherwise insoluble. The neutral chaotropic agent
urea is used at 5–9 M, often with up to 2 M thiourea,
which can dramatically increase the number of
proteins solubilized (Rabilloud et al. 1997). Thiourea
is weakly soluble in water but more soluble in high
concentrations of urea; therefore, a mixture of 2 M
thiourea and 5–8 M urea is used when strongly
chaotropic conditions are required. Charged
chaotropic agents such as guanidine hydrochloride
are incompatible with IEF.
Theory and Product Selection
If using thiourea during sample preparation, also add it
to the first-dimension rehydration solution; otherwise,
the proteins that require thiourea for solubility will
come out of solution during IEF.
Urea and thiourea can hydrolyze to cyanate and
thiocyanate, respectively; these products modify
amino groups on proteins (carbamylation) and give
rise to artifactual charge heterogeneity. Since heat
promotes this hydrolytic reaction, never heat ureaor thiourea-containing solutions above 37°C in the
presence of protein (McCarthy et al. 2003).
Detergents
Detergents disrupt hydrophobic interactions between
and within proteins and are classified as neutral,
zwitterionic, anionic, and cationic (Luche et al. 2003).
Some proteins, especially membrane proteins, require
detergents for solubilization during isolation and for
maintaining solubility during IEF.
Sample preparation for 2-D electrophoresis commonly
uses neutral or zwitterionic (having both positive and
negative charges resulting in a neutral net charge)
detergents at concentrations of 1–4%, since these
detergents do not introduce a net charge and therefore
allow proteins to migrate at their own charges during
IEF. Examples of neutral detergents include Tween,
octylglucoside, dodecyl maltoside, Triton X-100, and
Triton X-114. Examples of zwitterionic detergents
include CHAPS, CHAPSO, ASB-14, and SB 3-10.
In practice, only a few detergents are used in IEF
(Table 2.2). With few exceptions, only a single
detergent should be used because the effects of
detergents are not additive and can be unpredictable
in combination. Anionic and cationic detergents
are generally not suitable for IEF.
SDS is unparalleled in its ability to efficiently
and rapidly solubilize proteins. Although SDS is
incompatible with IEF as an anionic detergent, it can
be used in the initial preparation of concentrated
protein samples. In these cases, another IEFcompatible detergent must be used in excess to
disrupt the binding of SDS to protein (Ames and
Nikaido 1976). Also to be considered is how the
detergent interacts with high concentrations of
urea. When using SB 3-10, for example, the urea
concentration is limited to 5 M, but ASB-14 can be
used with 9 M urea (Chevallet et al. 1998).
15
Chapter 2: Sample Preparation
2-D Electrophoresis Guide
Reducing Agents
Reducing agents cleave disulfide bond crosslinks within and between protein subunits, thereby
promoting protein unfolding and maintaining proteins
in their fully reduced states. The compounds used
for 2-D sample preparation are either sulfhydryl or
phosphine reducing agents. Examples of sulfhydryl
reductants include dithiothreitol (DTT), dithioerythritol
(DTE), and b-mercaptoethanol (BME). DTT and
DTE can be used at lower concentrations than
b-mercaptoethanol and are more commonly used, but
high concentrations of DTT can affect the pH gradient
since its pKa is around 8. Examples of phosphine
reductants include tributylphosphine (TBP) and Triscarboxyethylphosphine (TCEP). These reducing agents
can be used at lower concentrations and over a wider
pH range than the sulfhydryl reductants; however,
their use is limited by low solubility and instability (TBP)
or a highly charged characteristic (TCEP).
Reducing agents added during protein extraction help
to solubilize proteins; during IEF, however, reducing
agents such as DTT become depleted from the basic
end of pH gradients extending above pH 8, which can
cause proteins to aggregate and precipitate (Hoving
et al. 2002). The result is streaking and other random
spot patterns, particularly in the alkaline regions of
the IPG strip (Herbert et al. 2001). To address this
problem, proteins can be reduced with TBP and then
irreversibly alkylated with iodoacetamide (Figure 2.1).
This treatment blocks protein sulfhydryls and prevents
proteins from aggregating and precipitating due to
oxidative cross-linking, ensuring that proteins remain
soluble throughout electrophoresis (Figure 2.2).
S
–S
–S
HS
–
–
S
SH
S
S
O
O
HS
–S
ReadyPrep reduction-alkylation kit
pH 3
pH 10
ReadyPrep Reduction-Alkylation Kit
O
SH
pH 10
Fig. 2.2. Effect of treatment with the ReadyPrep reduction-alkylation kit. Human HeLa cell extract (100 µg) separated by 2-D
electrophoresis (first dimension on 11 cm ReadyStrip™ IPG strips pH 3–10, second dimension using 12% Criterion™ gels) and stained with
Flamingo™ protein gel stain. The sample treated with the ReadyPrep reduction alkylation kit (right) and shows much better spot resolution
than the untreated sample (left), especially in the basic range of the gel.
H2N
S–
Untreated
pH 3
Theory and Product Selection
NH2
O
N
H2
NH2
Bio-Rad’s ReadyPrep reduction-alkylation kit
provides the reagents for reduction and alkylation of
sample proteins prior to IEF. Its use produces a 2-D
pattern with more spots, fewer streaks, and greater
reproducibility.
ReadyPrep Reduction-Alkylation Kit
Reduction cleaves disulfide bridges
and allows unfolding
Protein with disulfide bridges
Alkylation with iodoacetamide prevents
disulfide bridges from reforming
Ampholytes, Buffers, and Other Additives
Sample solution components that modify pH or
impart ionic strength affect the solubilization of
proteins during sample preparation and strongly
influence 2-D electrophoresis.
O
R1
S
S
Disulfide
R2
+
+
P
+
R1—SH
H2O
Tributylphosphine
R2—HS
+
Thiols
Tributylphosphine oxide
Reduction
O
R—SH
Thiol
+
I
P
Carrier ampholyte mixtures increase both buffering
power and ionic strength. Unlike non-ampholytic
ions, they do not interfere with IEF and can, in fact,
improve protein solubility by “salting in” proteins
that are otherwise insoluble under IEF conditions.
In addition, carrier ampholytes can diminish proteinmatrix interactions, which tend to occur at the basic
end of an IPG strip and lead to streaking caused
by precipitation (Righetti and Gianazza 1987).
Carrier ampholytes are routinely added to solutions
used during IEF with IPG strips and can be of value
during protein extraction as well.
Since proteins are often more soluble and proteases
are less active at higher pH, a base such as Tris may
be included in a lysis solution to elevate pH.
Many proteins also require ions in solution for optimum
solubility. Normally, this is achieved by adding salt
to the sample solution; however, adding salt prior to
IEF increases conductivity and consequently limits
the voltage at which IEF can be performed until the
salt is eventually removed from the system. Ions also
leave the IPG strip during IEF, causing any protein
requiring ions for solubility to precipitate. Proteins also
become less soluble as they approach their pI; they
may precipitate at their pI in a phenomenon known as
isoelectric precipitation or pI fallout.
O
R
NH2
Iodoacetamide
S
NH2
+
HI
Alkylated thiol
Alkylation
Fig. 2.1. Reduction and alkylation.
16
17
Chapter 2: Sample Preparation
2-D Electrophoresis Guide
Table 2.2. Summary of compounds used in 2-D electrophoresis sample solutions. Refer to Ordering Information (Appendix C) for catalog
numbers and details of options available for purchase.
Compound or product
Role in
Solution
Concentration
Range
Comments
Urea
Chaotrope
5–9.5 M
Present during first-dimension IEF
Thiourea
Chaotrope
2 MUsed with urea, usually in the combination 7 M urea, 2 M thiourea;
more effective than urea alone for solubilizing hydrophobic or high
molecular weight proteins
CHAPS
Detergent
1–4% (w/v)Zwitterionic detergent that may enhance protein solubility with minimal
disruptive effect on 2-D electrophoresis (Perdew et al. 1983)
CHAPSO
Detergent
1–4% (w/v)Zwitterionic detergent similar to CHAPS
NP-40
Detergent
0.5–1% (w/v)Neutral detergent originally used in 2-D electrophoresis (O’Farrell 1975,
Görg et al. 1988); its use has been largely superseded by CHAPS
(Görg et al. 2004)
Triton X-100
Detergent
0.5–1% (w/v)Neutral detergent similar to NP-40 also used for 2-D sample
preparation (Kawaguchi and Kuramitsu 1995)
SB 3-10
Detergent
1–2% (w/v)Zwitterionic detergent shown in some cases to give better solubilization
than CHAPS; insoluble in higher concentrations of urea and generally
used with 5 M urea, 2 M thiourea (Rabilloud et al. 1997)
ASB-14
Detergent
1–2% (w/v)Zwitterionic detergent developed for solubilization of membrane
proteins to be analyzed by 2-D electrophoresis (Chevallet et al. 1998)
ASB-C8Ø
Detergent
1–2% (w/v)Zwitterionic detergent developed for solubilization of membrane
proteins to be analyzed by 2-D electrophoresis (Chevallet et al. 1998)
Sodium dodecyl sulfate
Detergent
(SDS)
Up to 2% (w/v) Anionic detergent widely used in sample preparation for
during sample electrophoresis and unparalleled in its ability to solubilize protein;
preparation, no more also effective at inactivating proteases and other undesirable
than 0.2% (w/v) enzymatic activities. It is, however, incompatible with IEF unless during IEF diluted to 0.2% or less and used with at least an eightfold excess of
an IEF-compatible detergent such as CHAPS
Dithiothreitol (DTT)
Reductant
20–60 mMMost commonly used sulfhydryl reductant for 2-D electrophoresis
b-Mercaptoethanol
Reductant
1–5% (v/v)Sulfhydryl reductant originally used for 2-D electrophoresis (O’Farrell
1975); must be used at a relatively high concentration and can cause
disturbances to IEF, so is rarely used
Tributylphosphine (TBP)
Reductant2 mMPhosphine reductant effective at low concentrations and reported
to enhance solubilization of recalcitrant samples (Herbert et al.
1998). It has low water solubility and is unstable and therefore not
recommended as the sole reductant for first-dimension IEF
Tris-carboxyethylphosphine Reductant
2–40 mM
Phosphine reductant that may be useful during sample preparation;
(TCEP) it is highly charged and so is not recommended as the sole reductant
present during first-dimension IEF
18
Tris
Base
10–40 mM(Unbuffered) free base often added to sample preparation solutions to
raise the pH to a range where proteolysis is minimal and proteins are
optimally soluble. Other bases (for example, potassium carbonate or
spermine) are occasionally used as well (Rabilloud 1999). If Tris is used
during sample preparation, it should be diluted to 20 mM or less for
first-dimension IEF, as it may cause disturbances in the basic pH range
Bio-Lyte ® ampholytes
Carrier
0.2–1.0% (w/v)
Carrier ampholytes may be used during sample preparation to
ampholyteenhance protein solubility. Although IEF with IPG strips does not
require carrier ampholytes for pH gradient generation, the presence
of a relatively low (0.2% [w/v]) concentration of carrier ampholyte
is essential for optimum resolution. Use pH 3–10 ampholytes
or ampholytes appropriate to the IPG strip pH range
Removal of Interfering Substances
Impurities such as ionic detergents, lipids, nucleic
acids, salts and other ionic compounds, and
even high-abundance proteins can impact a 2-D
electrophoresis experiment by interfering with protein
separation or by obscuring proteins of interest.
These interfering substances can be endogenous
(for example, phenolics, lipids, and nucleic acids) or
exogenous (added during sample preparation; for
example, salts and detergents). Either way, removing
these impurities prior to analysis or mitigating their
effect is often essential for good results.
General Considerations
Though removal or mitigation of interfering
substances often yields clearer 2-D patterns and
improves resolution of protein spots, any treatment
of the sample can reduce yield and alter the relative
abundance of sample proteins. Procedures for
the removal of interfering substances represent
a compromise between removal of non-protein
contaminants and minimal interference with the
integrity and relative abundance of the sample
proteins. Since proteomics aims to study the
relationship among proteins in their natural state, it
is important to remove an interfering substance only
when necessary and by using techniques appropriate
for the sample.
Protein precipitation is a common general method
for contaminant removal. Conditions are chosen
under which sample proteins are selectively
precipitated while leaving soluble the major nonprotein contaminants. Following centrifugation, the
precipitated proteins are resuspended in a solution
suitable for IEF. Methods used in sample preparation
for 2-D electrophoresis include precipitation with
TCA and acetone (Damerval et al. 1986, Görg et al.
1988) and precipitation with methanol and chloroform
(Wessel and Flügge 1984). Precipitation procedures
also have the benefit of concentrating sample
protein, which is often necessary for effective
sample application.
Theory and Product Selection
Individual types of interfering contaminants cause
specific problems and can be removed or mitigated
in different ways. The most prevalent interfering
contaminants and their removal methods are
discussed next.
Nucleic Acids (DNA and RNA)
Nucleic acids, particularly DNA, can interfere with
IEF (for example by clogging gel pores) and increase
sample viscosity, thus limiting the effectiveness of cell
lysis and sample application. Because smaller nucleic
acids are generally tolerated better, strategies to
reduce nucleic acid interference involve either
shearing or enzymatic digestion: sonication shears
DNA and renders the sample less viscous, and
addition of nuclease digests nucleic acids to
oligo- or mononucleotides.
Nucleases are often employed during sample
preparation, particularly with bacterial lysates in
which nucleic acid:protein ratios are high. Successful
application of nuclease treatment requires attention
to three factors:
■■
■■
■■
ucleases may be inactive under the strongly
N
denaturing conditions often used to prepare protein
samples for 2-D electrophoresis
DNase requires magnesium ions for activity
ucleases are proteins and can appear in the
N
2-D pattern as extra spots
Benzonase is a nuclease with properties that make
it particularly useful in sample preparation for 2-D
electrophoresis (Chan et al. 2002). It is active in
the presence of urea, and the amount required for
treatment is usually not visible in a 2-D gel. It is
applied in the presence of 1 mM MgSO4 or MgCl2.
The magnesium ions are subsequently sequestered
with EDTA in order to inhibit proteases that may require
metal ions for activity.
19
Chapter 2: Sample Preparation
2-D Electrophoresis Guide
Polysaccharides
Lipids
Polysaccharides can interfere with electrophoresis by
clogging gel pores and by forming complexes with
proteins. Like nucleic acids, they can also cause a
sample to be viscous, making it difficult to work with.
Polysaccharides are a particularly prominent problem
with plant-derived samples.
Lipids can form insoluble complexes with proteins,
but lipids can also complex with detergents, thereby
reducing the detergents’ effectiveness at solublilizing
protein. The effect of lipids can be minimized by
using excess detergent (for example, 4% CHAPS in
the lysis solution when preparing lipid-rich tissues
such as brain). Precipitation methods that employ
organic solvents (Damerval et al. 1986, Görg et al.
1988, Wessel and Flügge 1984) or the ReadyPrep 2-D
cleanup kit can also be used to remove lipids.
Centrifugation may be used to remove high molecular
weight polysaccharides. Phenol extraction, followed
by precipitation with ammonium acetate in methanol,
is a commonly used method that is very effective
at removing polysaccharides in plant samples
(Hurkman and Tanaka 1986, Wang et al. 2008).
Phenolic Compounds
Phenolic compounds are found in all plants and
in some microorganisms and they can modify
proteins in an enzyme-catalyzed oxidative reaction.
The modification can cross-link proteins together or
render them insoluble. The reaction can be prevented
with reductants such as DTT, b-mercaptoethanol,
or ascorbic acid, and the enzyme is inactivated
by thiourea. Phenolic compounds may also be
removed from the extract using the ReadyPrep
2-D cleanup kit (see the Products for Contaminant
Removal sidebar) or by including polyvinylpyrrolidone
(PVP) or polyvinylpolypyrrolidone (PVPP) in the
extraction solution. These compounds bind phenolic
compounds, and the precipitated complex can
be removed from the extract by centrifugation
(Toth and Pavia 2001). The phenol extraction
procedure described above (see Polysaccharides)
is also effective at removing phenolic contaminants
(Hurkman and Tanaka 1986, Wang et al. 2008).
Before
Salts and Other Small Ionic Compounds
IEF requires samples that are free of salts and other
small ionic compounds that may interfere with pH
gradient formation. Salts formed from strong acids
and strong bases (for example, NaCl) dissociate into
their component base and acid, which is eventually
drawn to either end of the IPG strip. Until this occurs,
the conductivity of the IPG strip remains high and the
voltage attained is low. The flow of ions from the IPG
strip is accompanied by water flow, and one end of
the strip may dry out, breaking electrical contact.
Weak acids and weak bases (for example, acetate,
Tris, or ammonium ions) may not completely leave the
IPG strip during focusing. These compounds interfere
with the pH gradient, resulting in streaking and loss
of resolution at one end of the pH range or the other
(Figure 2.3). Amphoteric buffers such as HEPES can
focus within the pH gradient, resulting in a portion of
the pH gradient where proteins focus poorly.
Theory and Product Selection
Samples of low ionic strength are desired, yet many
samples contain salts and small ionic compounds that
are either intrinsic to the sample type or have been
introduced during sample preparation. Precipitation
and dialysis methods are very effective at removing
ionic contaminants, as is treatment with a desalting
column (Chan et al. 2002).
Prevention of Keratin Contamination
Skin keratin is a common contaminant of 2-D gels
and mass spectra. It may appear in silver-stained and
fluorescently stained 2-D gels as an artifact focusing
near pH 5 in the 50–70 kD region, or as an irregular
but distinctive vertical streaking parallel to the
SDS-PAGE direction of migration. The best remedy
for this keratin artifact is to avoid introducing it into the
sample in the first place. Filter all monomer solutions,
stock sample buffers, gel buffers, and electrode
buffers through nitrocellulose and store them in
sealed containers; then, clean the electrophoresis
cell thoroughly with detergent. Above all, careful
sample handling is important when sensitive detection
methods are used, and gloves should be worn while
handling samples, solution, or equipment.
Products for Contaminant Removal
For quick and effective contaminant removal,
Bio-Rad offers:
■■
After
■■
eadyPrep 2-D cleanup kit, which uses an
R
optimized version of a TCA-sodium deoxycholate
coprecipitation procedure (Arnold and UlbrichHoffmann 1999) to quantitatively precipitate
proteins while removing most interfering
substances. The protein precipitation process
also enables concentration of proteins from
samples that are too dilute, allowing for higher
protein loads that can improve spot detection
Bio-Spin®
ReadyPrep 2-D Cleanup Kit
Reservoir
Reservoir
Bio-Spin™
6 and Micro
6 columns
are ready to use and are filled with Bio-Gel®
P-6 support for the quick desalting and buffer
exchange of protein samples
End cap
End cap
3 cm
2 cm working bed height
5 cm
0.8 ml bed volume
3.7 cm working bed height
1.2 ml bed volume
Porous 30 µm
polyethylene bed
support retains
fine particles
Luer end fitting
with snap-off tip
Micro Bio-Spin Column
Luer end fitting
with snap-off tip
Fig. 2.3. Effect of salt removal. E. coli extracts containing 1 M NaCl were separated by 2-D electrophoresis before and after treatment with
the ReadyPrep 2-D cleanup kit. The samples were focused using 11 cm ReadyStrip pH 3–10 IPG strips and then separated on Criterion 8–16%
Tris-HCl precast gels.
20
Bio-Spin Column
21
Chapter 2: Sample Preparation
2-D Electrophoresis Guide
Prefractionation
Proteomic analysis is often applied to samples that
have undergone prior fractionation (prefractionation),
and the reasons for this are varied. In cases where
only a defined subset of the proteome is under
study, prefractionation can increase the chances
of meaningful discovery by removing proteins not
likely to be of interest from the sample. For example,
in studies of mitochondrial processes, it is sensible
to perform the proteomic analysis on a subcellular
fraction enriched in mitochondria. In other cases,
specific proteins of interest may be enriched through
fractionation and analyzed by 2-D electrophoresis
in the absence of potentially interfering proteins.
Prefractionation can also be used to separate a
sample into multiple fractions of lower complexity
that can then be analyzed separately; this can
enable identification of lower-abundance proteins
that might otherwise be undetectable in the
unfractionated sample.
Prefractionation increases the depth of proteome
analysis, but it does so at the expense of a greater
workload and reduced throughput. Try to use a
fractionation method that generates minimal protein
overlap between fractions.
Proteins can be fractionated by a number of different
techniques. The choice of method depends on
the sample, experimental goals, and available
instrumentation:
■■
■■
22
hemical and centrifugal methods —
C
use of selective precipitation or selective extraction
or centrifugation steps to separate proteins or
partition different subcellular compartments.
In many instances, protein extraction protocols
can incorporate fractionation steps through the
selective use of certain chemical reagents
lectrophoretic methods — application of
E
liquid-phase IEF or preparative SDS-PAGE with
the goal of protein enrichment. Though neither
of these techniques is orthogonal to either of the
two dimensions employed in 2-D electrophoresis
and neither offers additional resolving power to
the analysis, electrophoresis has proven useful in
allowing the enrichment of low-abundance proteins.
A protein in a size- or pI-enriched fraction can be
subjected to 2-D electrophoresis at a higher amount
relative to the unfractionated sample, allowing the
analysis of proteins present below detection levels
(Zuo and Speicher 2000, Fountoulakis and
Juranville 2003)
■■
hromatographic methods — use of
C
chromatographic separation principles to enrich
low-abundance proteins or generate fractions of
reduced complexity (Fountoulakis et al. 1997,
Badock et al. 2001, Butt et al. 2001, Smith et al.
2004, Qin et al. 2005, Yuan and Desiderio 2005).
Virtually any chromatographic procedure can be
used as a prefractionation step; examples include
size exclusion, affinity, ion exchange, and reversephase resins
Products for Fractionation by
Subcellular Location
■■
■■
Fractionation by Subcellular Location
Bio-Rad offers several ReadyPrep protein extraction
kits for the isolation of fractions enriched in integral
membrane and transmembrane proteins (Figure 2.4),
as well as nuclear and cytoplasmic proteins (see the
Products for Fractionation by Subcellular Location
sidebar).
larger numbers of transmembrane domains) are
better isolated using the membrane II kit, which
enriches integral membrane proteins by treating
a membrane preparation with sodium carbonate
(Fujiki et al. 1982, Molloy et al. 2000); this protocol
requires ultracentrifugation
Each of the following kits produces a fraction
with a distinct protein composition:
Using these methods alone or in combination, proteins
can be separated upstream of 2-D electrophoresis
(prefractionated) by their physical or chemical
properties, as described below. Some of these
methods, however, may introduce ionic or other
contaminants that must be removed before IEF.
Also, increasing the number of sample handling steps
may increase variability and the risk of sample loss.
There are many techniques for preparing fractions
enriched in subcellular organelles or membrane
types, and there are several examples in which these
techniques have been used to prepare samples for
2-D electrophoresis and other proteomic analyses
(Huber et al. 2003). Methods for organellar
fractionation generally involve differential and density
gradient centrifugation (Stasyk et al. 2007, Fialka et
al. 1997). However, fractionation schemes involving
aqueous polymer phase separation (Tang et al.
2008) and free-flow electrophoresis (Zischka et al.
2003, Eubel et al. 2008) have been described for this
purpose as well. These methods are usually specific
for the source material (cells or tissue). In some cases,
fractions representing different subcellular sites can
be generated on the basis of solubility under different
conditions (see the Fractionation by Solubility/
Hydrophobicity section). These methods are
more general in application.
Theory and Product Selection
A
eadyPrep protein extraction kit (signal) takes
R
advantage of the limited solubility of plasma
membrane microdomain structures (for example,
lipid rafts and caveolae) in nonionic detergents
at 4°C to yield a protein pellet that is enriched
in membrane-associated signalling proteins,
including glycophosphatidylinositol (GPI)-anchored
proteins, caveolin and associated proteins,
acetylated tyrosine kinases, and G proteins
(Simons and Ikonen 1997)
eadyPrep protein extraction kits (membrane I and
R
membrane II) use different techniques to isolate
integral membrane and membrane-associated
proteins without the need for density gradients.
The membrane I kit is based on temperaturedependent partitioning of hydrophobic proteins
into the detergent-rich phase of a Triton X-114/
water two-phase system (Bordier 1981, Prime
et al. 2000, Santoni et al. 2000). It is a quick
and effective protocol for enriching membrane
proteins without the need for ultracentrifugation.
More complex membrane proteins (those with
B
■■
eadyPrep protein extraction kit (cytoplasmic/
R
nuclear) uses a proprietary buffer and differential
centrifugation to isolate intact nuclei and a strongly
chaotropic extraction buffer to quickly prepare
highly enriched fractions of cytoplasmic and
nuclear proteins from eukaryotic samples
ReadyPrep Protein Extraction Kit
C
Fig. 2.4. Differences in 2-D patterns obtained using ReadyPrep protein extraction kits: signal (A), membrane I (B), and membrane II (C)
kits. Mouse liver samples were extracted using each kit, and purified proteins were separated using 17 cm ReadyStrip pH 3–10 NL IPG strips
and 8–16% gels. Overall spot patterns differ for A, B, and C even though all three kits isolate membrane proteins, indicating that each kit isolates
different sets of proteins.
23
Chapter 2: Sample Preparation
2-D Electrophoresis Guide
Products for Fractionation by
Solubility/Hydrophobicity
■■
■■
Products for Fractionation by
Protein Charge
For prefractionation in a convenient kit format,
Aurum™ AEX (anion exchange) and CEX (cation
exchange) mini kits and columns employ ion
exchange chromatography in an easy-to-use spin
column format for fractionating and concentrating
acidic and basic proteins from small sample volumes
(<1 ml). Micro Bio-Spin 6 columns are included for
salt removal from the fractionated samples.
Requiring only 15–20 min operating time, Aurum
ion exchange mini spin columns provide a quick,
convenient, and reproducible sample preparation
tool for 2-D electrophoresis, and their use can
improve detection of low-abundance proteins
(Liu and Paulus 2008).
eadyPrep sequential extraction kit is based on
R
a published method (Molloy et al. 1998) that uses
sequentially more highly solubilizing chaotrope
and detergent mixtures. Applying each extracted
fraction to a separate gel allows the resolution of
more protein spots
eadyPrep protein extraction kit (soluble/insoluble)
R
uses a different set of detergents to fractionate
proteins on the basis of their solubility in detergents
The ReadyPrep sequential extraction kit and the
ReadyPrep protein extraction kit (soluble/insoluble)
can be used either independently or sequentially for
even greater depth of coverage.
ReadyPrep
reagent 1
Protein
sample
Step 1
Insoluble pellet
from reagent 1
Collect supernatant 1
Reagent 2
Theory and Product Selection
ReadyPrep
reagent 2
ReadyPrep Sequential Extraction Kit
Step 2
Insoluble pellet
from reagent 2
ReadyPrep
reagent 3
Collect supernatant 2
Step 3
Insoluble pellet
from reagent 3
Total Protein
pH 3
pH 10
AEX bound fraction
pH 3
Aurum Ion Exchange Kit
pH 10
AEX unbound fraction
pH 3
pH 10
Collect supernatant 3
Fig. 2.6. Fractionation of rat brain tissue using Aurum ion exchange mini columns. Rat brain total protein extracts (3 ml) were loaded onto
an Aurum AEX column and eluted. The unfractionated and fractionated samples were then treated with the ReadyPrep reduction alkylation
and 2-D cleanup kits and separated by 2-D electrophoresis. Red circles indicate a group of protein spots with increased intensities after
fractionation. Blue arrows show two representative spots detected only in the gels of the AEX bound fraction.
Fractionation by pI
Fig. 2.5. Distribution of proteins based on differential solubility using the ReadyPrep sequential extraction kit. The generation of three
fractions provides increased resolution of proteins on 2-D gels.
Fractionation by Solubility/Hydrophobicity
Fractionation by Protein Charge
Proteins can be separated according to their
solubility in different reagents using either chemical
or chromatographic methods. Sequential extraction
under different solvent conditions can be used to
fractionate a protein sample based on solubility,
and this strategy has also been used to prepare
discrete fractions for analysis by 2-D electrophoresis
(Lenstra and Bloemendal 1983, Weiss et al. 1992).
Extraction using different detergents can also
yield different protein fractions (Figure 2.5), and
chromatographic methods that can be used include
reverse-phase (Van den bergh and Arckens 2008)
and hydrophobic interaction chromatography
(McNulty and Annan 2009).
Ion exchange chromatography has been used to
reduce proteome complexity, enrich low-abundance
proteins, and improve peptide mass fingerprints
(Butt et al. 2001). This technique separates proteins
according to their charge at various pHs. It is based
on the reversible adsorption of proteins to a solid
phase containing charged chemical groups.
Cationic (+) or anionic (-) resins (Figure 2.6) attract
molecules of opposite charge in the solvent. A variety
of systems and media are available for ion exchange
chromatography, but because elution involves gradient
elution by washing the column with buffers of gradually
increasing ionic strength or pH, a subsequent
cleanup step must be included.
Fractionation by pI, for example by liquid-phase IEF,
may seem counterintuitive as a fractionation technique
upstream of the first-dimension IEF separation. It can,
however, improve downstream sample loading and
separation on narrow- and micro-range IPG strips by
eliminating proteins outside the pH region of interest
(Figure 2.7). This unique separation method can also
be coupled to analytical or preparative SDS-PAGE for
a powerful, complementary first-dimension separation
and enrichment strategy for high molecular weight,
membrane, hydrophobic, or other proteins that
are often underrepresented in IPG-based 2-D gels
(Davidsson 2002, Hansson et al. 2004, Brobey
and Soong 2007)2.
2
24
iquid IEF introduces ampholytes that must be removed, for example
L
with the ReadyPrep 2-D cleanup kit, before IEF in IPG strips.
25
Chapter 2: Sample Preparation
2-D Electrophoresis Guide
Fractionation by Size (MW)
Products for Fractionation by pI
The Rotofor®, Mini Rotofor, and MicroRotofor cells
separate and concentrate proteins into discrete
fractions by liquid-phase IEF. Following ampholyte
removal and sample concentration with the
ReadyPrep 2-D cleanup kit, each of the resulting
liquid fractions can then be separated on narrowor micro-range IPG strips.
Size-dependent separation is a powerful fractionation
strategy in studies focused on a particular protein or
protein family and their posttranslational modifications
because these proteins tend to be of similar size
(Fountoulakis and Juranville 2003). Proteins can
be separated into size-dependent fractions by
polyacrylamide gel electrophoresis (PAGE), particularly
continuous-elution electrophoresis.
Products for Fractionation by Size (MW)
The Model 491 prep cell and mini prep cell
perform size-dependent high-resolution
fractionation of proteins by continuous-elution gel
electrophoresis (using native PAGE or SDS-PAGE).
The large sample capacity (50 µl–15 ml, and
0.5–500 mg protein) of these cells makes them
particularly effective tools for the enrichment of
low-abundance proteins (Zerefos et al. 2006,
Xixi et al. 2006, Fountoulakis et al. 2004).
Rotofor Family of Liquid-Phase IEF Cells
Unfractionated
pH 3
10
10
Before
One of the major difficulties facing proteomics is the
issue of dynamic range, or the variation in abundance
among sample proteins that typically spans several
orders of magnitude. This range typically exceeds that
over which proteins can be effectively detected and
quantified. Various strategies have been developed
for the reduction of sample dynamic range, and
they have proven beneficial for the study of lowabundance proteins.
Depletion
Samples may be dominated by a few abundant
proteins whose presence can obscure less abundant
proteins and limit the capacity and resolution of the
separation technique employed. This is particularly
apparent for serum and plasma; the study of lowerabundance proteins from serum or plasma is
often complicated by the presence of albumin and
immunoglobulin G (IgG), which together contribute
up to 90% of the total protein in a serum sample.
These proteins obscure comigrating proteins and limit
the amount of total serum protein that can be loaded
on 2-D gels. To obtain meaningful results from serum
samples, these proteins must be removed (Figure 2.8).
A strategy for specific depletion of abundant proteins
by immunoaffinity chromatography has been widely
used (Pieper et al. 2003, Roche et al. 2009, Tu et al.
2010, Ichibangase et al. 2008). Though this method is
effective, the need for antibodies renders it expensive
and limits its applicability to the specific sample type
for which the antibodies were developed.
Fraction 3, pH 6.04
pH 3
Depletion and Dynamic Range Reduction
Theory and Product Selection
Albumin
Heavy-chain IgG
Light-chain IgG
After
Albumin
Fig. 2.8. Albumin and IgG removal from serum using the Aurum
serum protein mini kit. Serum proteins were separated by 2-D
electrophoresis before and after treatment with an Aurum serum
protein mini column. Albumin and IgG are removed following treatment
with the column, improving resolution of other protein species.
Samples (100 µg) were focused on 11 cm ReadyStrip pH 5–8 IPG
strips, then run on 8–16% gels.
Products for Depletion
Fraction 3, pH 6.04
pH 4.7
5.9
Model 491 Prep Cell and Mini Prep Cell
Bio-Rad’s Aurum Affi-Gel® Blue and Aurum serum
protein mini kits represent a simple, low-cost
alternative to immunodepletion. These kits use
affinity chromatography to easily and effectively
remove albumin (Affi-Gel Blue) or albumin and IgG
(serum protein kit) in a single spin column.
Aurum Ion Exchange Kit
Fig. 2.7. Clean fractionation by pI. Mouse liver extract was
fractionated using the MicroRotofor cell. 2-D separations of the
unfractionated sample (120 µg) and fractions (30 µg) are shown.
Prior to 2-D separation, samples were treated with the ReadyPrep
2-D cleanup kit to remove extra ampholytes. Note the clean pH
boundaries of fraction 3 and the enrichment of proteins in the pH
region it covers.
26
27
Chapter 2: Sample Preparation
2-D Electrophoresis Guide
Dynamic Range Reduction
Untreated
ProteoMiner™
protein enrichment technology uses a
bead-based library of combinatorial peptide ligands
that act as unique binders for proteins (Thalusiraman
et al. 2005, Guerrier et al. 2006). When a complex
sample is applied to the beads, abundant proteins
saturate their specific ligands while the remaining
proteins can be washed away. Low-abundance
proteins are concentrated on their specific ligands
and will be enriched relative to the abundant proteins
following elution. In contrast to immunodepletion,
ProteoMiner has no intrinsic specificity for any
particular sample type and can be used to decrease
high-abundance proteins in any sample that could
benefit from such a treatment. The technology has
been most widely applied to serum and plasma
(Sennels et al. 2007); however, several examples
of successful application of ProteoMiner to other
samples have also been reported (Castagna et al.
2005, Guerrier et al. 2007, D’Ambrosio et al. 2008,
Bandhakavi et al. 2009).
Theory and Product Selection
ProteoMiner Technology
ProteoMiner technology employs a combinatorial
library of hexapeptides bound to a chromatographic
support. Combinatorial synthesis creates a large library
of unique hexapeptides, with each hexapeptide bound
to a stationary support, or bead. Each bead, featuring
a unique ligand, is expected to bind specifically to one
or a small number of different proteins in a mixture,
and the library of all possible sequences binds proteins
up to the capacity of available beads.
Treated
When a complex biological sample is applied to the
beads, high-abundance proteins saturate their ligands
and excess proteins are washed away. In contrast,
low-abundance proteins do not saturate their binding
sites. Therefore, different samples retain relative
expression levels similar to the original samples.
Moreover, low-abundance proteins are enriched if the
beads are eluted in a volume smaller than the original
sample. The overall effect of ProteoMiner technology
results in the bound and eluted material consisting of a
significantly lower amount of total protein, thus allowing
resolution of a greater diversity of species.
Biological sample
(large dynamic range)
ProteoMiner protein enrichment kits:
■■
■■
■■
ecrease the amount of high-abundance proteins
D
without immunodepletion, preventing the loss of
proteins bound to high-abundance proteins
nrich medium- and low-abundance proteins that
E
cannot be detected through traditional methods
(Figure 2.9)
o not rely on a predefined set of antibodies,
D
unlike immunodepletion products
■■
Are compatible with a variety of sample types
■■
Offer a convenient, easy-to-use format
■■
Can be used for differential expression analysis
Bind mixture
to library
Wash away
unbound protein
Elute bound
sample for analysis
Fig. 2.9. The ProteoMiner protein enrichment kit improves
resolution and spot counts in 2-D gels. In an untreated sample,
albumin and other high-abundance proteins dominate the gel and
obscure signals from less abundant proteins. On a gel generated
using an equal amount of total protein from a treated serum sample,
however, resolution is dramatically improved and a greater number
of protein spots is visualized.
Ligand library
Depletion of plasma and serum samples by ProteoMiner technology. Each bead features a different hexapeptide ligand with affinity
for specific proteins in a sample. Samples are applied to the beads, allowing proteins to bind to their specific ligands. Proteins in excess are
washed away, and those proteins bound to the beads are eventually eluted, allowing further downstream analysis.
28
29
Chapter 2: Sample Preparation
2-D Electrophoresis Guide
Additional Resources
Samples can be prepared for 2-D electrophoresis
using many other techniques. Consult Posch (2008)
for more information on:
■■
■■
■■
■■
■■
■■
The chemical components of the sample buffer and
the amount of protein available for assay dictate the
type of assay that may be used.
■■
ample preparation basics (cell disruption, sample
S
solubilization, protein assays, contaminant removal)
Protein labeling techniques
ractionation using chemical reagents
F
and chromatography
Fractionation using electrophoresis methods
nrichment strategies for organelles, multiprotein
E
complexes, and specific protein classes
pplication of sample preparation tools and
A
fractionation strategies to study different
biological systems
Sample Quantitation (Protein Assays)
Determine the concentration of protein in a sample
(Berkelman 2008) by protein assay to:
■■
■■
■■
nsure that the amount of protein to be
E
separated is appropriate for the IPG strip length
and visualization method
acilitate comparison among similar samples;
F
image-based analysis is simplified when equivalent
quantities of proteins have been separated
The most commonly used protein assays are
visible assays, assays in which the presence of protein
causes a visible color change that can be measured
with a spectrophotometer (Sapan et al. 1999;
Noble and Bailey 2009; see the Protein Assay
Products and SmartSpec™ Plus Spectrophotometer
sidebar). All protein assays utilize a dilution series of a
known protein (usually bovine serum albumin or bovine
g-globulin) to create a standard curve from which the
concentration of the sample is derived (for a protocol
describing protein quantitation, refer to Part II of
this guide).
■■
radford assays (Bradford 1976) — based on an
B
absorbance shift of Coomassie (Brilliant) Blue G-250
dye under acid conditions, when a redder form of
the dye is converted into a bluer form upon binding
to protein. The increase of absorbance at 595 nm
is proportional to the amount of bound dye and,
therefore, to the amount (concentration) of protein
present in the sample. In comparison to other protein
assays, the Bradford protein assay is less susceptible
to interference by various chemicals that may be
present in protein samples, with the exception of
elevated concentrations of detergents like SDS.
The response of the Bradford protein assay is
only slightly affected by urea, thiourea, and
CHAPS in concentrations up to 1.75 M, 0.5 M,
and 1% (w/v), respectively
owry (Lowry et al. 1951) — combines the
L
reactions of cupric ions with the peptide bonds
under alkaline conditions with the oxidation of
aromatic protein residues. The Lowry method is
based on the reaction of Cu+, produced by the
peptide-mediated reduction of Cu2+, with FolinCiocalteu reagent (a mixture of phosphotungstic acid
and phosphomolybdic acid in the Folin-Ciocalteu
reaction). The Lowry assay is intolerant of thiourea,
reductants such as DTT, and chelating agents
such as EDTA
CA (bicinchoninic acid, Smith et al. 1985) —
B
BCA reacts directly with Cu+ (generated by peptidemediated reduction of Cu2+) to produce a purple end
product. The reagent is fairly stable under alkaline
conditions and can be included in the copper
solution to allow a one-step procedure. Like the
Lowry assay, the BCA assay is intolerant of thiourea,
reductants such as DTT, and chelating agents such
as EDTA
2-D sample solutions typically contain reagents
that interfere with all of the assays described above.
The Bradford assay may be used on samples that
are concentrated enough to be diluted with water
so that urea, thiourea, and CHAPS are no longer
present at interfering levels (typically at least fourfold).
Otherwise, modified assay procedures may need to
be employed (see the Protein Assay Products sidebar).
30
Theory and Product Selection
Protein Assay Products
Protein concentration in 2-D sample solutions
is best measured using the RC DC™ protein
assay, a modification of the Lowry assay that
incorporates a precipitation step that removes
reducing agents and detergents. For more
information on protein quantitation using visible
assays, refer to Bio-Rad bulletin 1069.
Table 2.3. Bio-Rad protein assay selection guide.
Quick Start™ Bradford
Bio-Rad
DC™
RC DC
Method
Bradford
Lowry
•
—
•
—
—
•
—
•
Description
One-step determination;
not for use with
SDS-containing samples
Standard Bradford assay, not to be
used with elevated
levels of detergents
(>0.1% SDS)
Detergent compatible
(DC); Lowry assay
modified to save time
and to be more
accurate
Reducing agent
and detergent
compatible (RC DC)
Standard-concentration Assay
Sample volume
Linear range
100 µl
0.125–1.5 mg/ml
100 µl
0.125–1.5 mg/ml
100 µl
0.125–1.5 mg/ml
100 µl
0.2–1.5 mg/ml
Low-concentration Assay
Sample volume
Linear range
Microplate assay volume
Minimum incubation
Assay wavelength
1 ml
1.25–25 µg/ml
5 µl
5 min
595 nm
800 µl
1.25–25 µg/ml
10 µl
5 min
595 nm
200 µl
5–250 µg/ml
5 µl
15 min
650–750 nm 200 µl
5–250 µg/ml
**
15 min
650–750 nm
SmartSpec Plus Spectrophotometer
The color change observed in protein assays is
measured using a spectrophotometer. Bio-Rad’s
SmartSpec Plus spectrophotometer has
preprogrammed methods for protein quantitation
and a working wavelength range of 200–800 nm.
It can be used for routine applications such as:
■■
uantitation of proteins via the Bradford,
Q
Lowry, and BCA assay methods
■■
Quantitation of DNA, RNA, and oligonucleotides
■■
Monitoring bacterial culture growth
■■
Simple kinetic assays
■■
Wavelength scans with peak detection
SmartSpec Plus Spectrophotometer
Features built into the SmartSpec assay
methods facilitate data collection and present
a complete analysis of assay results. Bio-Rad
also offers compatible quartz and UV-transparent
plastic cuvettes.
31
2-D Electrophoresis Guide
Theory and Product Selection
CHAPTER 3
The First Dimension:
Isoelectric Focusing
(IEF)
32
33
Chapter 3: The First Dimension: Isoelectric Focusing (IEF)
2-D Electrophoresis Guide
Protein Separation by Isoelectric point (pI)
The first-dimension separation of 2-D electrophoresis
is IEF, where proteins are separated on the basis of
differences in their pI. The pI of a protein is the pH at
which it carries no net charge, and it is a characteristic
that is determined by the number and types of
charged groups the protein carries.
uncharged and stops migrating (Figure 3.2).
If, by diffusion, it drifts away from the point in the
gradient corresponding to its pI, it acquires charge
and is pulled back. In this way, proteins condense,
or are focused, into sharp bands in the pH gradient
at their characteristic pI values.
IEF proceeds until a steady state is reached.
Proteins approach their pI values at different rates
Proteins are amphoteric molecules, which carry a
but remain relatively fixed at those pH values for
positive, negative, or zero net charge depending on
extended periods. This is in contrast to conventional
the pH of their environment. For every protein, there
electrophoresis (for example, polyacrylamide gel
is a specific pH at which its net charge is zero (its pI).
electrophoresis, or PAGE), where proteins continue
Proteins show considerable variation in pI, though pI
to move through the medium until the electric field is
values usually fall in the range of pH 3–12, with the
removed. Moreover, in IEF, proteins migrate to their
majority falling between pH 4 and pH 8. A protein
COO
COO
COOH
steady-state
positions from
anywhere in the system.
is positively charged at pH values below its pI and
2-D electrophoresis is performed underNH
negatively charged at pH values above its pI (Figure 3.1). IEF for NH
NH
denaturing conditions so that proteins are completely
For IEF, a protein is placed in a medium with a pH
disaggregated and all charged groups are exposed
gradient and subjected to an electric field. In response
COO
COO
COOH
to the bulk solution. Consequently, resolution is best
to the field, the protein moves toward the electrode
under denaturing conditions. Complete denaturation
NH
with the opposite charge. Along the way, it either
NH
NH
and solubilization are required to minimize aggregation
picks up or loses protons. Its net charge and mobility
and intermolecular
thus ensuring
that
pH interactions,
= pl
pH > pl
decrease until the protein eventually arrives at the pointpH < pl
each protein is present in only one configuration.
in the pH gradient equal to its pI. There, the protein is
3
3
COO
COOH
COO
2
3
Net Charge
NH3
NH2
COOH
COO
COO
Isoelectric point (pl)
+1
pH < pl
3
NH2
NH3
4
5
6
7
8
9
10
11
pH
–1
pH = pl
pH > pl
–2
–3
Fig. 3.1. Net
Dependence
of protein net charge on the pH of its environment. The pH at which the net charge is 0 is the isoelectric point (pI).
Charge
+3
+2
9
Anode
+
5
4
+1
3
0
36
4
4
8
8
5
3
6
7
78
9
10
11
5
7
6
Isoelectric
point (pl)
5
10
pH
9
4
10
6
7
3
9
10
6
8
4
5
3
3
4
5
6
–2
7
Anode
+
3
3
pH
3
5
4
4
4
4
4
5
5
5
5
6
8
9
9
77
8
6
7
8
6
7
8
6 6
Supplied commercially and ready to use
■■
Prepared on a plastic backing to simplify handling
■■
■■
■■
Carrier ampholyte tube gels must be cast by the user
arrier ampholyte–generated pH gradients drift
C
over time and are, therefore, not as reproducible
as immobilized pH gradients
arrier ampholytes are complex chemical
C
mixtures, and batch-to-batch variations affect the
characteristics of the pH gradient
arrow pH gradients and gradients encompassing
N
the extremes of the pH range (below pH 4 and above
pH 9) cannot be accommodated
Tube gels can be difficult to handle
ighly reproducible and stable over even extended
H
IEF runs (Bjellqvist et al. 1982)
vailable in a wide variety of pH gradients and
A
lengths (see the ReadyStrip™ IPG Strips sidebar)
8
10
9
9
10
9
9
10
10
Relative separation. Relative focusing power
expresses the enhanced resolution expected in the
first dimension when using IPG strips of different
lengths or pH ranges. The 7 cm pH 3–10 IPG strip
Cathode
–
10
Fig. 3.2. Principle of IEF. A mixture of proteins is separated in a pH gradient and within an electric field according to each protein’s pI
and independently of its size. The proteins migrate until they reach their pI.
34
■■
■■
■■
Strip Range* Focusing
3
■■
is arbitrarily assigned a baseline focusing power of
1.0 to calculate the relative focusing powers of the
other strips.
ReadyStrip IPG strips are preprinted to indicate anode (+) and pH range;
in addition, a bar code is printed on the 24 cm strip.
ReadyStrip IPG strip pH ranges.
9
–3
3
■■
Cathode
–
–1
pH
using carrier ampholyte–generated pH gradients
and tube gels. This type of first dimension has been
largely superseded by the use of IPG strips for the
following reasons:
IPG strips are:
IPG strips simplify first-dimension separations by
immobilizing the pH gradient on an easy-to-handle
support strip. ReadyStrip IPG strips are available
in a wide selection of pH gradients and strip
lengths (from 7 to 24 cm) to fit Bio-Rad vertical
electrophoresis cells and gels. Premade ReadyStrip
IEF buffers are also available for convenience and
maximum reproducibility.
+2
0
NH3
IEF for 2-D electrophoresis is most commonly
performed using immobilized pH gradient (IPG) strips.
As their name implies, IPG strips contain buffering
groups covalently bound to a polyacrylamide gel strip
to generate an immobilized pH gradient. The pH
gradients are created with sets of acrylamido buffers,
which are derivatives of acrylamide containing
both reactive double bonds and buffering groups.
The general structure is CH2=CH–CO–NH–R, where
R contains either a carboxyl [–COOH] or a tertiary
amino group (for example, –N(CH3)2). These acrylamide
derivatives are covalently incorporated into
polyacrylamide gels at the time of casting and
can form almost any pH gradient (Righetti 1990).
ReadyStrip IPG Strips
+3
NH3
IEF Media: IPG Strips vs. Carrier Ampholytes Historically, first-dimension IEF was performed
2
3
Theory and Product Selection
pH 345678910
Relative Focusing Power
7 cm11 cm17 cm18 cm24 cm
ReadyStrip IEF Buffer
3–10 7–10 3.9–5.14.7–5.95.5–6.76.3–8.3
Broad Range
3–10
3–10 nonlinear (NL)
1×
1×
1.6×
1.6×
2.4× 2.6× 3.4×
2.4× 2.6× 3.4×
Narrow range
3–6
5–8
7–10
4–7
2.3×
2.3×
2.3×
2.3×
3.7×
3.7×
3.7×
3.7×
5.7×
5.7×
5.7×
5.7×
Micro range
3.9–5.1
4.7–5.9
5.5– 6.7
6.3– 8.3
5.8×
5.8×
5.8×
3.5×
9.2× 14.2× 15.0× 20.0×
•
9.2× 14.2× 15.0× 20.0×
•
9.2× 14.2× 15.0× 20.0×
•
5.5× 8.5× 9.0× 12.0×
6.0×
6.0×
6.0×
6.0×
•
•
8.0×
•
8.0×
•
8.0×
8.0×
•
•
•
* Strips are designed with sufficient overlap to allow spot matching while limiting the extent of redundant data.
35
Chapter 3: The First Dimension: Isoelectric Focusing (IEF)
2-D Electrophoresis Guide
■■
Choice of pH Gradient
IPG strips are available in various pH gradients
(see the table in the ReadyStrip IPG Strips sidebar).
The pH gradients are linear (pH varies in a linear
manner with respect to length of the strip) except in
the case of nonlinear pH 3–10 gradients (NL, see the
Estimation of pI sidebar).
■■
■■
se broad-range strips (for example, pH 3–10)
U
for an overview of the spot distribution along the
pH gradient and for comparing different sample
preparation strategies. Since many proteins focus
se narrow- and micro-range gradients for greater
U
resolution (there is a larger separation distance, more
cm of gel, per pH unit). With the exclusion of proteins
outside the pH range of the strip, more total protein
mass can be loaded per strip to also allow detection
of more proteins
se overlapping pH ranges to increase resolution by
U
expanding a small pH range across the entire width
of a gel (Figure 3.3). This also allows the creation
of composite gels by matching spots from the
overlapping regions using imaging software
pH 3–10
Estimation of pI
The pI of a protein is a useful parameter for protein
characterization. With purified proteins, pI can be
determined by IEF using IPG strips, usually under
denaturing conditions. Using linear IPG strips, the pH
gradient can be assumed to extend linearly between
the pH extremes. Knowing the length and pH range
of the IPG strip implies that experimental pI values can
be assigned with a high level of accuracy (see figure).
Protein pI estimations can also be made using NL
IPG strips, assuming the pH profile of the IPG strip is
available from the manufacturer; without the exact pH
profile of the strip, the pI estimate will be less accurate.
For pI estimation, stain the IPG strips after IEF,
for example with Bio-Safe Coomassie blue stain,
and then plot the migration distance along the length
of the IPG strips of each of the protein standards.
Graph A shows the pH gradient along the length
of a linear pH 4–7 IPG strip. To determine the pI of
an unknown, simply determine the band position
(as a percentage of gel length) and read the pI from
the graph. In the example, a band positioned at 50%
of the gel length will have an estimated pI of 5.5.
The same strategy can be applied for protein spots
on 2-D gels, but with less accuracy due to swelling
or shrinkage of the 2-D gel. It may also be difficult to
define the start and end positions of the IPG strip on
stained 2-D gels.
pH 3–6
pH 5–8
pH 7–10
Fig. 3.3. A mouse liver sample was extracted in a urea-thiourea-CHAPS solution. The extract was run in a single PROTEAN® i12™ IEF cell
run on twelve 11 cm ReadyStrip IPG strips simultaneously at each of the following pH ranges: 3–10, 3–6, 5–8, and 7–10. Each pH gradient was
run in triplicate. The second dimension for each IPG strip was run in 8–16% gradient Criterion™ precast gels that were stained with Bio-Safe™
Coomassie stain. The above figure shows a representative gel image for each pH range.
Choice of IPG Strip Length
■■
IPG strips are available in a variety of lengths that
match the size of most commercial second-dimension
vertical electrophoresis systems. Shorter strips match
mini-format systems, and longer strips match largeformat systems. Deciding which strips to use depends
on the requirements for speed, sample volume,
resolution, and throughput (see Chapter 4 for more
details on selecting size format for 2-D electrophoresis):
■■
■■
■■
36
se shorter strips and mini-format gels for
U
fast, convenient sample screening or method
development
se longer strips for the best separation with
U
higher protein loads and for maximum resolution.
The longest IPG strips and large-format gels have
a large area to resolve protein spots; however, they
take a long time to run
ombine different size formats for various benefits.
C
For example, use a mini-format system for rapid
optimization of sample preparation methods, then
switch to a large format for thorough assessment of
a complex sample and identification of proteins of
interest. In many cases, a mini system and narrowrange IPG strips can then be used to focus in on
proteins of interest
A. Linear pH 4–7 ReadyStrip IPG strip
7
6
pH
When selecting the IPG strip, consider both the pH
gradient and strip length, as both determine the
resolution in the final 2-D gel (see the ReadyStrip IPG
strips sidebar).
in the middle of the pH range 3–10, using NL
gradients can improve resolution of proteins in the
middle of that range and compress the width of the
extreme pH ranges at the ends of the gradients
5
4
0
25
50
75
100
75
100
B. Nonlinear pH 3–10 ReadyStrip IPG strip
10
9
8
pH
Selection of IPG Strips
Theory and Product Selection
7
6
5
4
3
0
25
50
% Total IPG strip length
Estimating the pI of a protein from its position along an IPG
strip. A, By plotting the pH of an IPG strip as a function of its
length, the pI of a protein may be derived from its focused position
on that strip. In the example shown, the pI of a protein that migrates
across 50% of the strip length is 5.5. B, pH profile of Bio-Rad
ReadyStrip nonlinear pH 3–10 IPG strips.
With knowledge of experimental pI and molecular
weight values (see Chapter 4 for details about
molecular weight estimation), it is possible to make
comparisons with the calculated values derived after
protein spot identification using mass spectrometry.
The calculation of theoretical pI values is possible with
software tools available on the Internet, for example
at http://web.expasy.org/compute_pi. If the values
differ significantly from each other, this may indicate
a false identification or the identification of a fragment
of the respective protein. However, differences in pI or
molecular weight can also suggest posttranslational
modifications, such as phosphorylation or glycosylation.
The detection of posttranslational modifications
is a unique strength of gel-based proteomics.
These modifications offer information about the
function, regulation, and cellular location of proteins.
se overlapping narrow- and micro-range IPG strips
U
to increase the effective length of pI resolution.
When three narrow-range overlapping ReadyStrip
IPG strips (pH 3–6, 5–8, 7–10) are used with the
Criterion system, for example, the resolution in the
first dimension (11 cm strip, pH 3–10 NL) is increased
from 11 to 26 cm. When four micro-range strips
are used, the resolution in the first dimension is
expanded from 11 to 44 cm
37
Chapter 3: The First Dimension: Isoelectric Focusing (IEF)
2-D Electrophoresis Guide
Sample Application
Commercial IPG strips are dehydrated and must be
rehydrated to their original gel thickness (0.5 mm)
before use. The protein sample can be applied
to the IPG strip either during or after rehydration,
and rehydration can be done in either disposable
rehydration/equilibration trays or directly in the
focusing tray. Sample application during rehydration
is the easiest and, in most cases, most efficient way to
apply sample. In some instances, however, it is best
to rehydrate the IPG strips and then apply sample
through sample cups while current is applied
(cup loading) (Table 3.1). Each method is discussed
in the following sections.
The rehydration solution generally contains the
following components to maintain protein solubility and
allow tracking of the separation (see Part II, Methods
for recipes):
■■
■■
■■
■■
■■
Chaotrope — urea (8 M or up to 9.8 M if necessary
for sample solubility), with or without 2 M thiourea
he composition of the rehydration solution should
T
also resemble the composition of the sample solution
in terms of the additives present to aid solubility. If the
sample was prepared using thiourea, the rehydration
solution should also contain thiourea. Likewise, the
same detergent should be used. Otherwise, transition
from one solution to the other can cause precipitation
of proteins that are soluble in the sample solution but
not in the rehydration solution
Sample Application during Rehydration
In this option, the sample is prepared in, or diluted into,
rehydration solution and introduced to the IPG strip
at the time of rehydration. As the strips hydrate for
at least 12 hr, proteins in the sample are absorbed
and distributed over the entire length of the strip
(Rabilloud et al. 1994, Sanchez et al. 1997).
Sample application can be in either the absence
(passive) or presence (active) of applied voltage:
■■
Detergent — nonionic or zwitterionic detergent such
as CHAPS, Triton X-100, or NP-40 at 0.5–4% (w/v)
Reducing agent — 20–100 mM DTT
Ampholytes — 0.2% (w/v), usually pH 3–10;
concentrations up to 1% (w/v) may be used,
though this reduces the voltage and results in
correspondingly longer runs
■■
Tracking dye — a trace of bromophenol blue to
render the IPG strip more visible for simplified handling
and act as a tracking dye for confirmation of focusing
ctive rehydration is performed in the IEF cell. A low
A
voltage (30–100 V) is applied, and proteins enter the
gel matrix under current as well as by absorption.
Active rehydration with the sample is believed to
promote the entry of large proteins into the strip by
applying an electrical “pull”
In passive rehydration, proteins enter the gel
by absorption only. This allows efficient use of
equipment, since strips can be rehydrated in sample
rehydration trays while other samples are focused in
the IEF cell
Whether IPG strips are rehydrated actively or passively,
they must be incubated with sample for at least 12 hr
prior to IEF. This gives high molecular weight proteins
time to enter the gel after the gel has become fully
hydrated and the pores have attained full size.
Sample application during rehydration works
because IEF is a steady-state technique; therefore,
proteins migrate to their pI independently of their initial
positions. The advantages of these approaches
over cup loading are:
■■
■■
■■
■■
Disadvantages
In-gel rehydration
Simple sample application
Poor resolution of basic proteins
■■
horter focusing times can be used because the
S
sample proteins are in the IPG strip prior to IEF
Large amounts of protein can be loaded
■■
up loading requires gel-side up strip placement so
C
that the sample cup may be placed in contact with
the gel surface
In-gel sample loading is conducted gel-side down.
If the IEF cell is programmed for an unattended start
following rehydration, IEF must be conducted gelside down as well
If in-gel sample loading is performed in the
rehydration/equilibration tray, IEF may be performed
either gel-side up or gel-side down. This is largely
a matter of user preference, though improved
resolution may be observed with the gel-side up
configuration, particularly with higher protein loads
■■
hen running micro-range IPG strips spanning
W
~1 pH unit
or samples that contain large amounts
F
of glycoproteins
No precipitation at the point of sample application
Accommodates dilute samples and larger protein loads
Passive
Focusing can follow rehydration without manual intervention if performed within the IEF instrument
Not all proteins, particularly large or hydrophobic
proteins, will be taken up
Active
More effective with certain proteins, particularly those of high molecular weight
Rehydration must occur within the IEF instrument
Cup loading
■■
In addition, electrode wicks may be placed between
the electrode in the focusing tray and the IPG strip in
To apply samples after IPG strip rehydration,
either running configuration. Electrode wicks serve
the sample is loaded into sample cups positioned
as a sink for ionic sample contaminants and proteins
on the rehydrated strip. This technique can be more
with pIs outside the pH range of the IPG strip used.
challenging than in-gel sample loading from a technical
They also prevent drying of the ends of the IPG
standpoint, but it can be beneficial in the following
strips during IEF. In some cases, however, the use of
cases (Cordwell et al. 1997, Görg et al. 2000):
electrode wicks has little effect on separation quality,
■■ When samples contain high levels of DNA, RNA,
and they may be omitted for convenience in either
or other large molecules, such as cellulose
running configuration if satisfactory results are
■■ When running acidic and basic IPG strips;
obtained in their absence.
for example, pH 7–10
Table 3.1. Advantages and disadvantages of sample loading methods.
Advantages
For IEF, the rehydrated IPG strips are placed into the
focusing tray. The orientation (gel-side up or gel-side
down) of the IPG strip in the focusing tray is largely
determined by the sample loading method employed:
Sample Application by Cup Loading
■■
Method
Setup for IEF
Simplicity
educed risk of sample precipitation, which can
R
occur with cup loading at the sample application
point if sample concentration is too high
(Rabilloud 1999)
Theory and Product Selection
More effective for basic proteinsSetup more complicated; the cup must form a seal
with the IPG strip
Can improve resolution at extremes of the pH gradient High protein loads are difficult to accommodate;
(the end opposite the point of application)concentrated samples are required.
Sample precipitation may occur at the point
of application
38
39
Chapter 3: The First Dimension: Isoelectric Focusing (IEF)
2-D Electrophoresis Guide
Power Conditions for IEF
The pH gradient and the electric field strength both
influence the time required to reach steady state and
the resolution of the separation. The electric field
strength is determined by the length of the IPG strip
and the applied electrical field. In general, narrow
pH ranges yield higher resolution and require higher
voltages and more time to reach a steady state.
Longer IPG strips can withstand higher programmed
voltages and require an increased number of volthours (Vh) for proper focusing. Vh are the product of
voltage and time. Because the actual voltage reached
is current dependent and the maximum programmed
voltage may not be reached, programming the IEF
cell with Vh can better ensure that samples receive
a consistent number of volts.
The electrical conductivity of the system changes
during an IEF run. At the beginning, the current is
relatively high because of the large number of charge
carriers present. As the proteins and ampholytes
move toward their pIs, and as ionic contaminants
move to the ends of the IPG strip, the current
gradually decreases. When the proteins reach their
final positions in the pH gradient, there is little ionic
movement in the system; the voltage reaches a
maximum, and the current reaches a minimum.
Focusing should occur with a gradual increase in
voltage followed by a prolonged focusing phase at the
maximum voltage advisable for the IPG strip length
used and until a set number of Vh have accumulated.
The optimum duration depends on the length of the
IPG strip and the pH gradient. Current is generally
limited to 50 µA per IPG strip, and a streamlined onestep protocol is adequate in most circumstances,
as the voltage will rise gradually without the need for
a phased focusing protocol with programmed voltage
ramping. A more gradual focusing protocol may be
advisable in circumstances of heavy protein load,
for some narrow- and micro-range IPG strips,
or when there are high levels of charged contaminants
present. Since the duration of the prolonged focusing
phase is specified in Vh, the actual duration may vary
depending on the average voltage during focusing.
Focusing may conclude at different times for IPG
strips run at the same time with the same protocol.
It is important, therefore, to include a hold step during
which the IPG strip is held at a relatively low voltage to
maintain focusing until the IPG strip can be removed
from the instrument.
Theory and Product Selection
PROTEAN i12 IEF System
The PROTEAN i12 IEF system allows multistep runs
at durations set by either time or Vh. Recommended
starting electrical conditions and voltage ramping
options are provided in Part II of this guide; however,
sometimes the number of Vh required to complete a
run must be determined empirically in a time course
experiment. The optimum Vh depends on the
sample and the composition of the sample solution
as well as the pH gradient of the IPG strip. A more
complex sample or different sample buffer might
change the Vh required. The time needed to achieve
the programmed Vh depends on the pH range
of the IPG strip as well as sample and buffer
characteristics.
Other IEF cells only support running identical pH
gradients and similar samples in batches because
the programmed current and voltage are spread
across all lanes. If different pH ranges or samples
with varying conductivity are run at the same time,
the electrical conditions experienced by individual
IPG strips are different. This may expose some
strips to more or less current than desired, since
the total current limit is averaged over the tray.
The individual lane control provided by the
PROTEAN i12 IEF cell, however, ensures that
the current limit is not exceeded in any IPG strip,
even in situations where conductivity differs
significantly among samples run at the same time.
The PROTEAN i12 IEF cell also allows each lane
to be programmed individually, making it possible
to run different protocols in different lanes.
The flexibility of this system allows running different
experiments at once or varying conditions within
an experiment to allow optimization in fewer runs.
The system also results in better reproducibility
because focusing conditions are not influenced
by other samples in the run.
Electrical current generates heat, which limits the
magnitude of the electric field that can be applied and
the ionic strength of the solutions that can be used.
Thin gels dissipate heat better than thick gels and thus
better withstand the high voltage that offers higher
resolution. Also, as mentioned above, the current
drops to a constant low value as focusing reaches
a steady state.
PROTEAN i12 IEF Cell and Accessories
40
41
2-D Electrophoresis Guide
Theory and Product Selection
CHAPTER 4
The Second Dimension:
SDS-PAGE
42
43
Chapter 4: The Second Dimension: SDS-PAGE
2-D Electrophoresis Guide
Theory and Product Selection
Mini-PROTEAN TGX
TGX
Mini-PROTEAN
Protein Separation by Size
Selection of Polyacrylamide Gels
The second-dimension separation is by protein size
(mass) using SDS-PAGE. The proteins separated in
IPG strips by IEF in the first dimension are applied to
polyacrylamide gels and separated a second time by
SDS-PAGE (Figure 4.1).
Polyacrylamide gels are prepared by free radical
polymerization of acylamide and a comonomer
crosslinker such as bis-acrylamide. By convention,
gels are characterized by two parameters that
determine pore size: total monomer concentration
(%T, in g/100 ml) and weight percentage of crosslinker
(%C). SDS-PAGE gels typically have a %C of 2.7%,
and the %T is varied to give separation characteristics
appropriate to the experimental needs. %T determines
the relative pore size of the resulting polyacrylamide
gel, with higher %T resulting in smaller pores and
separation characteristics more appropriate for
smaller proteins.
A two-step equilibration process prepares the proteins
for SDS-PAGE. The proteins are complexed with
SDS, reduced with DTT, and then alkylated with
iodoacetamide. Treatment of the proteins with SDS
yields protein-SDS complexes with a consistent
charge-to-mass ratio. When electrophoresed through
a polyacrylamide gel, these complexes migrate with a
mobility that is related logarithmically to mass. As the
proteins migrate through the gel, the pores of the gel
sieve proteins according to size.
Choice of Gel Percentage (Composition)
Gels are either purchased as commercial precast
gels or cast in the laboratory using unpolymerized
monomer and buffer components. Precast gels are
available in smaller formats to fit commercially available
electrophoresis cells. These are appropriate for the
second dimension when the first dimension is run on
7 cm or 11 cm IPG strips. Larger second dimensions
are generally run on lab-cast gels.
Gels for SDS-PAGE are made with either a single,
continuous %T throughout the gel (single-percentage
gels) or a gradient of %T (gradient gels). Gradient gels
are cast with acrylamide concentrations that increase
from top to bottom so that the pore size decreases
as proteins migrate into the gels. Single percentage
gels are cast in the laboratory by simply pouring the
appropriate percentage of acrylamide, along with
bis-acrylamide, buffer, initiator, and catalyst, into a
gel cassette prepared using glass plates and spacers
clamped together. The mixture is poured into the
cassette and allowed to polymerize. A stacking layer
is not necessary for second-dimension gels. Gradient
gels may also be cast in the laboratory using solutions
of differing acrylamide percentage and a gradient
maker. Typical gel compositions are 7.5–20% for
single-percentage gels, and 4–15% to 10–20% for
gradient gels.
Use protein migration charts and tables to select
the gel type that offers optimum separation of your
sample (Figure 4.2):
■■
First Dimension
Isoelectric focusing (IEF),
separation by pl
Low pH
Second Dimension
SDS-PAGE,
separation by MW
High pH
■■
High MW
■■
se single-percentage gels to separate bands of
U
similar size. Since optimum separation occurs in the
lower half of the gel, choose a percentage in which
the protein of interest migrates to the lower half of
the gel
se gradient gels to separate a broad range of
U
protein sizes. Gradient gels allow resolution of
both high- and low-molecular weight bands on the
same gel. The larger pore size towards the top of
the gel permits resolution of larger molecules, and
decreasing pore sizes toward the bottom of the gel
restrict excessive separation of small molecules.
Gradient gels are often the most appropriate choice
for 2-D electrophoresis, which is most often applied
to complex samples with proteins spanning a large
size range
or new or unknown samples, use a broad gradient
F
gel (for example, 4–20 %T or 8–16 %T or Bio-Rad’s
Any kD™ 3 formulation) for a global evaluation of the
sample, and then move to an appropriate singlepercentage gel for more detailed investigation of a
particular size range of interest
Precision Plus Protein Unstained
Precision Plus Protein Unstained
7.5%
7.5%
250
250
150
150
10%
10%
250
250
150
150
100
100
100
100
75
75
12%
12%
250
250
150
150
250
250
100
100
150
150
75
75
50
50
75
75
50
50
37
37
7.5%
7.5%
200
200
116
116
50
50
37
37
25
25
20
20
10%
10%
200
200
116
116
97.4
97.4
97.4
97.4
66
66
66
66
45
45
4–15%
4–15%
37
37
25
25
20
20
15
15
100
100
75
75
50
50
31
31
31
31
21.5
21.5
250
250
150
150
100
100
75
75
50
50
37
37
37
37
25
25
20
20
15
15
10
10
25
25
20
20
15
15
10
10
Broad Range Unstained
Broad Range Unstained
12%
4–15%
4–20%
12%
4–15%
4–20%
200
200
200
200
116
116
97.4
97.4
116
116
66
66
97.4
97.4
45
45
66
66
31
31
200
200
116
116
97.4
97.4
66
66
45
45
Any kD
Any kD
250
250
150
150
100
100
75
75
50
50
37
37
25
25
20
20
15
15
10
10
Any kD
Any kD
200
200
116
116
97.4
97.4
66
66
45
45
31
31
45
45
31
31
31
31
45
45
4–20%
4–20%
21.5
21.5
21.5
21.5
14.4
14.4
14.4
14.4
6.5
6.5
21.5
21.5
21.5
21.5
14.4
14.4
6.5
6.5
14.4
14.4
6.5
6.5
Fig. 4.2. Examples of migration charts. The protein standards
were run on Mini-PROTEAN® TGX™ gels.
Low MW
Fig. 4.1. Separation of proteins by SDS-PAGE after separation by IEF. The IPG strip containing proteins already separated by pI is applied
to the top of a polyacrylamide gel. The proteins are then separated according to size (MW) by SDS-PAGE.
3
44
io-Rad’s Any kD formulation provides separation of 10–250 kD
B
proteins, with the best resolution in to the 20–100 kD range.
These gels are useful for screening samples or for 2-D applications
aimed at rapid protein analysis.
45
Chapter 4: The Second Dimension: SDS-PAGE
2-D Electrophoresis Guide
Choice of Gel Size
Vertical Electrophoresis Systems
for SDS-PAGE
The electrophoresis systems offered by Bio-Rad
for second-dimension electrophoresis are detailed
in the table.
■■
he Mini-PROTEAN system includes the
T
Mini-PROTEAN Tetra cell (with a capacity
of up to four gels) and the high-throughput
Mini-PROTEAN® 3 Dodeca™ cell (for running up to
12 gels). These systems accommodate 7 cm IPG
strips and are compatible with handcast or precast
Mini-PROTEAN gels (8.6 × 6.7 cm)
The Criterion™ system includes the Criterion
cell (for 1–2 gels) and the high-throughput
Criterion Dodeca cell (for 1–12 gels); both cells
accommodate Criterion precast gels (13.3 × 8.7 cm)
and 11 cm IPG strips
■■
he PROTEAN II and PROTEAN Plus Dodeca
T
systems accommodate 17, 18, or 24 cm IPG strips.
The PROTEAN II system provides the ability to
choose the glass plates, spacer, and sandwich
clamps to cast two gel lengths: 16 or 20 cm.
The PROTEAN Plus Dodeca cell allows maximum
throughput for 2-D electrophoresis, with the
capability to run up to 12 2-D gels at a time
■■
Bio-Rad’s vertical electrophoresis systems.
The choice of gel size depends on the same factors
determining the length of IPG strip used, namely
speed, resolution, and throughput (see the Choice of
IPG Strip Length section):
■■
■■
■■
ini-format systems accommodate short IPG strips
M
(7 cm) and mini-format gels. The short separation
distance of the gels maximizes the electrical field
strength (V/cm) to yield rapid separations with
moderate resolution. Use mini gels and systems for
rapid analysis and method development
idi gels and midi-format systems accommodate
M
11 cm IPG strips and are slightly larger (both in
width and length) gels. They still offer rapid runs,
but because of the longer separation distance,
they provide better coverage than mini-format gels
arge-format systems accommodate 17–24 cm
L
IPG strips and large gels and offer the maximum
resolution possible; however, large gel sizes require
longer run times
Choice of Buffer System
Mini-PROTEAN System
Criterion System
PROTEAN II System
PROTEAN Plus System
Cells
Mini-PROTEAN Tetra Cell
Criterion Cell
PROTEAN II XL Cell
PROTEAN Plus Dodeca Cell
Mini-PROTEAN Dodeca Cell
Criterion Dodeca Cell
PROTEAN II XL Multi Cell
Number of gels
1–4
1–2
1–4*
1–12
1–6
PROTEAN II handcast
PROTEAN Plus handcast
17 cm
18 and 24 cm
1–12
Gel formats
Mini-PROTEAN precast
Criterion precast Mini-PROTEAN handcast
Criterion empty cassettes
IPG strip length
7 cm
11 cm
1–12
* For 2-D applications running a maximum of 2 gels at a time is recommended.
PowerPac™ Power Supplies
Power supplies are required to meet the power
requirements of numerous applications. The choice
of power supply for second-dimension PAGE usually
depends on the size and number of gels being run:
■■
■■
■■
46
se the PowerPac Basic or PowerPac HC highU
current power supply for mini-format vertical
PAGE applications
PowerPac HC High-Current
Power Supply
PowerPac Basic
Power Supply
PowerPac Universal
Power Supply
PowerPac HV High-Voltage
Power Supply
The pH and ionic composition of the buffer systems
used to prepare the gels and samples and to fill the
electrode reservoirs determine the power requirements
and heavily influence the separation characteristics of
a polyacrylamide gel. Different buffer systems also
vary widely in their stability.
Theory and Product Selection
The most common buffer system for seconddimension SDS-PAGE is the Tris-HCl system
described by Laemmli (Laemmli 1970). The reagents
are inexpensive and readily available, and the precast
gels are also readily available in a wide variety of gel
percentages. The system is robust and highly tolerant
of high sample loads. However, Tris-HCl resolving
gels are prepared at pH 8.6–8.8; at this basic pH,
polyacrylamide slowly hydrolyzes to polyacrylic acid,
which can compromise separation. For this reason,
Tris-HCl gels have a relatively short shelf life.
In addition, the gel pH can rise to pH 9.5 during a
run, causing proteins to undergo deamination
and alkylation, thereby diminishing resolution and
complicating post-electrophoresis analysis.
To alleviate these shortcomings, a number of
alternative buffer systems have been developed.
For example, bis-Tris, Tris-acetate, and other
proprietary buffer systems (see the Precast Gels for
Second-Dimension SDS-PAGE sidebar) offer extended
shelf life as well as other separation characteristics
unique to their formulations.
High-quality precast gels are preferred for highthroughput applications. They provide savings in time
and labor, and the precision-poured gradients result in
reproducibility among runs.
Precast Gels for Second-Dimension
SDS-PAGE
Bio-Rad offers precast gels in two size formats and
in a variety of formulations, some of which feature
IPG wells to hold two lengths of ReadyStrip™ IPG
strips (7 cm and 11 cm).
Bio-Rad’s TGX™ (Tris-Glycine eXtended shelf life)
precast gels are Laemmli gels with a proprietary
modification that extends shelf life to 12 months
and allows gels to be run at higher voltages without
producing excess heat. The TGX formulation does
not require special, expensive buffers. Like Tris-HCl
gels, TGX gels use a discontinuous buffer system,
with glycinate as the trailing ion, and are, therefore,
compatible with conventional Laemmli and
Tris/glycine/SDS buffers. These are the best
choice when long shelf life is needed and
traditional Laemmli separation patterns are desired.
TGX Stain-Free™ gels are Laemmli-like extended
shelf life gels that allow rapid fluorescent detection
of proteins with the stain-free enabled imagers
Gel Doc™ EZ and ChemiDoc™ MP, eliminating
staining and destaining steps. Other precast
gel formulations have also been developed to
circumvent the shelf life issues of Tris-HCl systems.
se the PowerPac HV high-voltage or PowerPac
U
Universal power supply for large-format vertical
PAGE applications
se the PowerPac HC power supply for
U
applications that require high currents, such as
PAGE with the high-throughput Dodeca cells
47
Chapter 4: The Second Dimension: SDS-PAGE
2-D Electrophoresis Guide
Theory and Product Selection
Precast Gels for Second-Dimension SDS-PAGE (contd.)
Gel Format and Formulation Selection Criteria
Composition
Gel Format and Formulation Selection Criteria
Composition
Migration (%T)
Mini-PROTEAN*
(for 7 cm IPG Strips)
Migration (%T)
Criterion*
(for 11 cm IPG Strips)
Mini-PROTEAN TGX Laemmli-like extended
Stain-Free
shelf life gels
Best choice when
long shelf life is
needed and traditional
Laemmli separation
patterns are desired
Stain-Free formulation
includes an additive
for rapid fluorescence
detection without
staining
Any kD
7.5%
10%
12%
Any kD
250
150
100
7.5%
250
150
75
50
10%
250
150
Criterion TGX
Laemmli-like extended
shelf life gels
Best choice when long shelf life is needed and traditional
Laemmli separation patterns are desired
12%
250
150
100
100
100
Any kD
7.5%
10%
12%
18%
4–15%
4–20%
8–16%
10–20%
75
100
75
7.5%
10%
250
250
150
150
100
75
37
50
50
37
25
25
37
10
Any kD
7.5%
250
150
75
50
250
150
100
75
75
50
50
37
75
37
250
50
20
50
20
10%
250
150
12%
100
150
150
100
75
100
75
50
75
50
4-20%
250
250
75
37
4-15%
250
150
100
100
10
15
37
50
25
75
50
37
37
20
37
25
20
50
15
37
10
20
25
20
25
15
15
25
20
Criterion TGX
Laemmli-like extended
Stain-Free
shelf life gels
Best choice when long shelf life is needed and traditional
Laemmli separation patterns are desired
Stain-Free formulation
includes an additive
for rapid fluorescence
detection without
staining
Any kD
7.5%
10%
12%
18%
4–15%
4–20%
8–16%
10–20%
Any kD
250
150
100
75
25
25
20
15
7.5%
10%
250
250
150
150
100
75
50
37
100
20
75
50
10
15
12%
18%
250
150
100
75
75
50
50
37
4–15%
250
25
100
20
75
25
20
25
20
15
10
15
25
20
15
37
37
25
20
20
15
15
10
10
10
4 –20% 8 –16% 10 –20%
250
50
10
15
250
150
100
75
75
75
50
50
37
37
25
37
20
25
25
20
25
20
15
10
250
150
100
100
15
20
10
25
50
37
50
50
37
150
150
37
15
75
20
25
15
250
150
100
50
50
75
75
10
37
75
250
150
100
25
20
10
250
150
100
100
15
100
37
25
250
150
150
25
25
20
20
15
25
4 –20% 8 –16% 10 –20%
50
37
25
15
100
250
150
100
4–15%
20
37
250
150
18%
37
20
Any kD
7.5%
10%
12%
4–15%
4–20%
12%
100
37
75
50
Mini-PROTEAN TGX Laemmli-like extended
shelf life gels
Best choice when long shelf life is needed and traditional
Laemmli separation
patterns are desired
250
150
50
75
37
Any kD
15
20
15
15
10
10
10
10
10
Mini-PROTEAN
Tris-Tricine
Best choice for
separation of low
MW proteins
16.5%
10–20%
16.5% 10–20%
25
20
25
15
20
15
10
5
10
Criterion Tris-HCl Reagents are easy to prepare, inexpensive,
and readily available
Best choice when switching between precast and handcast gels and need to
compare results 5%
7.5%
10%
12.5%
15%
18%
4–15%
4–20%
8–16%
10–20%
10.5–14%
5%
10%
250
250
150
150
12.5%
250
150
100
100
250
150
100
75
75
50
25
75
25
50
37
75
50
37
25
250
150
15
100
75
75
250
150
100
75
250
150
100
75
50
50
37
37
50
25
37
50
37
50
37
25
20
15
25
20
15
10
8–16% 10 –20% 10.5–14%
100
15
20
15
250
150
25
10
20
4–20%
250
150
100
75
5
100
4–15%
250
150
100
75
20
37
37
18%
50
37
50
2
15%
100
75
250
150
2
7.5%
10
25
20
20
15
15
10
10
10
25
20
15
10
* All gel percentages listed in bold are available in IPG and/or prep-well comb format.
48
49
Chapter 4: The Second Dimension: SDS-PAGE
2-D Electrophoresis Guide
Theory and Product Selection
Precast gels for Second-Dimension SDS-PAGE (contd.)
Gel Format and Formulation Selection Criteria
Composition
Gel Format and Formulation Selection Criteria
Composition
Migration (%T)
Criterion Stain Free
Tris-HCl
Reagents are easy to
prepare, inexpensive
and readily available
Migration (%T)
Criterion*
(for 11 cm IPG Strips)
Criterion*
(for 11 cm IPG Strips)
10%
4–20%
8–16%
Best choice when
switching between
precast and handcast
gels and when
comparing results
Stain-Free formulation
includes an additive
for rapid fluorescence
detection without
staining
10%
250
150
100
75
4–20%
250
Criterion Tris-Tricine
8–16%
16.5%
10–20%
16.5% 10–20%
250
150
100
150
75
26.6
26.6
100
75
17.0
50
14.0
50
50
37
Best choice for separation of low MW proteins
37
17.0
14.0
37
25
25
25
20
20
15
6.5
6.5
3.5
20
3.5
1.4
1.4
15
10
10
15
Criterion XT Bis-Tris Offer long shelf life, 10%
but require dedicated 12%
sample and running
4–12%
buffers
10%
12%
4–12%
Size, kD
(XT MES running buffer)
Criterion XT
Tris-Acetate
Offer best resolution of 7%
high molecular weight 3–8%
proteins, but require
dedicated sample and
running buffers
7%
10%
12%
4–12%
Size, kD
(XT MOPS running buffer)
3–8%
Size, kD
(XT Tricine running buffer)
* All gel percentages listed in bold are available in IPG and/or prep-well comb format.
50
51
Chapter 4: The Second Dimension: SDS-PAGE
2-D Electrophoresis Guide
Molecular Weight (MW) Estimation
■■
efore assembling the casting chamber, glass
B
plates should be carefully cleaned with Bio-Rad
cleaning concentrate and thoroughly rinsed with
deionized water
■■
■■
■■
ach pair of glass plates (two per gel) should
E
be separated from the next by a spacer sheet;
the spacer sheet allows easier separation of the
cassettes after gel polymerization
he volume of gel solution should be determined
T
by measuring the volume of water needed to fill the
assembled glass plates to the desired level in the
multi-casting stand
llow overnight polymerization to compensate for
A
the low concentrations of catalysts (recommended
to ensure that polymerization does not start while
the gradient gels are being cast)
Apparatus for casting multiple gels. Multi-casting chambers for 12 PROTEAN Plus™ gels or for 12 Mini-PROTEAN gels allow uniform casting
of gradient gels. Gradient makers are available for both size formats.
Transition from First to Second Dimension
The transition from first- to second-dimension gel
electrophoresis involves the following:
■■
■■
quilibration, which involves two steps that treat
E
the focused IPG strips with an SDS-containing
buffer to prepare the proteins for SDS-PAGE.
The first equilibration solution contains buffer,
urea, glycerol, reductant, SDS, and dye (optional).
The second equilibration step replaces the
reductant with iodoacetamide to alkylate the
thiol groups. Equilibration ensures the proteins
are coated with dodecyl sulfate and all cysteines
are reduced and alkylated
mbedding of the strip on the top of the secondE
dimension gel. The equilibrated IPG strips are placed
on top of the gel and sealed in place with molten
agarose solution to ensure good contact between
the gel and the IPG strip
Power Conditions and Reagents
for SDS-PAGE
For SDS-PAGE, use running conditions that provide
optimum separation across the size range of interest
and that maintain the temperature of the system
during operation. For a complete discussion of running
conditions and the parameters that affect them, please
refer to A Guide to Polyacrylamide Gel Electrophoresis
and Detection, Bio-Rad bulletin 6040. For seconddimension SDS-PAGE, include a short, low voltage
(50 V) step at the beginning of the run to ensure that all
of the proteins are removed from the IPG strip before
final voltages are applied.
SDS-PAGE is a reliable method for estimating the
MW of an unknown protein. The migration rate of a
protein–SDS complex is inversely proportional to the
logarithm of its MW: the larger the polypeptide, the
more slowly it migrates in a gel. The key to accurate
MW determination is selecting separation conditions
that produce a linear relationship between log (MW)
and migration within the likely MW range of the
unknown protein. These parameters are discussed
more thoroughly in Molecular Weight Determination
by SDS-PAGE (bulletin 3133), and a protocol for MW
estimation is provided in Part II of this guide.
For best results, separate the protein sample
on the same gel with a set of protein standards.
See The Little Book of Standards (bulletin 2414) and
the Protein Standards Application Guide (bulletin 2998)
for more information regarding selection of protein
standards. Mixtures of standard proteins with known
MW can be unstained, prestained, or include tags
for development with various secondary reagents
(useful when blotting). Standards can be run in a
reference well or attached to the end of a focused IPG
strip by filter paper onto the second-dimension gel.
For convenience, Bio-Rad’s Precision Plus Protein
standard plugs (catalog #161-0378), which are
embedded in agarose plugs, can also be used.
After separation, determine the relative migration
distance (Rf ) of the protein standards and of the
unknown protein. Rf is defined as the mobility of
a protein divided by the mobility of the ion front
(Figure 1). Because the ion front can be difficult to
locate, mobilities are normalized to the tracking dye
that migrates only slightly behind the ion front:
Rf = (distance to band)/(distance to dye front)
Using the values obtained for the protein standards,
plot a graph of log (MW) vs. Rf (see below). The plot
should be linear for most proteins, provided they
are fully denatured and that the gel percentage
is appropriate for the MW range of the sample.
The standard curve is sigmoid at extreme MW values,
because the sieving affect of the matrix is so large at
high MW that molecules are unable to penetrate the
gel; but at low MW, the sieving effect is negligible and
proteins migrate almost freely. To determine the MW
of the unknown protein band, interpolate the value
from this graph (Figure 2).
Gradient SDS-PAGE gels can also be used to estimate
MW. In this case, log (MW) is proportional to log
(%T). With linear gradients, %T is proportional to the
distance migrated, so the data can be plotted as log
(MW) vs. log (migration distance). Standard curves are
actually sigmoid. The apparent linearity of a standard
curve may not cover the full MW range for a given
protein mixture in a particular gel. However, log (MW)
varies sufficiently slowly to allow fairly accurate MW
estimates to be made by interpolation, and even
extrapolation, over relatively wide ranges.
12 34 5678
Top of resolving gel
MW, kD
Migration
distance of
unknown
band
(45 mm)
250
150
100
75
50
Migration
distance
of dye front
(67 mm)
37
25
20
Unknown band
15
10
Dye front
Fig. 1. Example showing MW determination of an unknown
protein. Lane 1, 10 μl of Precision Plus Protein unstained standards;
lanes 2–8, a dilution series of an E. coli lysate containing a
hypothetical unknown protein (GFP). Proteins were separated by
SDS-PAGE in a Criterion 4–20% Tris-HCI gel and stained with
Bio-Safe Coomassie stain.
3.0
log MW
Casting SDS-PAGE Gels Using
Multi-Casting Chambers
In general, proteomics work requires running
several IPG strips and second-dimension gels
per experiment. It is important that gels have a
very similar composition. The best way to ensure
that handcast gels have the same composition is
to cast them at the same time in a multi-casting
chamber. This is especially important when casting
gradient gels. Details of the assembly and use
of multi-casting chambers are available in their
accompanying instruction manuals. Tips that
generally apply to all multi-casting systems are:
Theory and Product Selection
Standards
Unknown
2.0
y = –1.9944x + 2.7824
r 2 = 0.997
1.0
0
0
0.2
0.4
0.6
0.8
1.0
Rf
Fig. 2. Determining the MW of an unknown protein by SDS-PAGE.
A standard curve of the log (MW) versus Rf was generated using the
Precision Plus Protein standards from Figure 1. The strong linear
relationship (r2 > 0.99) between the proteins’ MW and migration
distance demonstrates exceptional reliability in predicting MW.
Methods for equilibrating and embedding IPG strips
onto second-dimension gels are available in Part II
of this guide.
52
53
2-D Electrophoresis Guide
Theory and Product Selection
CHAPTER 5
Detection
54
55
Chapter 5: Detection
2-D Electrophoresis Guide
Detection of Proteins in Gels
In 2-D electrophoresis, proteins in gels are most
commonly visualized using total protein stains.
Selection of the most appropriate stain involves
consideration of the stain characteristics, limitations
with regard to the sensitivity of detection and the types
of proteins it stains best, downstream applications,
and the type of imaging equipment available
(see Chapter 6). For use in proteomics applications,
stains should be compatible with high-throughput
protocols and downstream analysis, including
digestion and mass spectrometry (Patton 2000).
It is also possible to label protein samples after sample
preparation and prior to IEF with fluorescent dyes such
as the CyDye DIGE fluors (Westermeier and Scheibe
2008). At the time of writing, three dyes with spectrally
different excitation and emission wavelengths were
available, allowing labeling of up to three different
samples and their separation in a single 2-D gel.
The dyes are matched for size and charge to obtain
migration of differently labeled identical proteins to the
same spot positions. The labeled samples are mixed
together before they are applied on the gel of the first
dimension. After separation, the gels are scanned with
fluorescence imagers at the different wavelengths.
The following are general tips for staining 2-D gels:
■■
■■
■■
56
-D gels are clearer, sharper, and more reproducible
2
when less protein is loaded. When sample
preparation and IEF conditions are not optimized,
it is often beneficial to load relatively little protein and
to use a relatively sensitive staining technique
o identify low-abundance proteins, apply a high
T
protein load and use a high-sensitivity stain
(for example, silver or a fluorescent stain)
(Corthals et al. 2000)
o obtain enough protein for mass spectrometry,
T
apply a high protein load and use a compatible
staining procedure
■■
■■
or quantitative comparisons, use stains with broad
F
linear ranges of quantitation (for example, Flamingo™,
Oriole™, and SYPRO Ruby)
ince no stain is capable of staining all proteins,
S
consider staining replicate gels with two or more
different stains. Coomassie (Brilliant) Blue appears
to stain the broadest spectrum of proteins.
Therefore, it is instructive, especially with 2-D gels,
to stain a Coomassie Blue–stained gel with silver,
or to stain a fluorescently stained gel with colloidal
Coomassie Blue or silver. Often, this double staining
procedure reveals a few differences between the
protein patterns. It is possible to stain gels first with
Coomassie Blue or a fluorescent stain, then again
with silver
The sensitivity achievable in staining is determined by:
■■
The amount of stain that binds to the proteins
■■
The intensity of the coloration
■■
he difference in color intensity between stained
T
proteins and the residual background in the body
of the gel (the signal-to-noise ratio); unbound stain
molecules can be washed out of the gels without
removing much stain from the proteins
No stain interacts with all proteins in a gel in precise
proportion to their mass, and all stains interact
differently with different proteins (Carroll et al. 2000).
The only observation that seems to apply for most
stains is that they interact best with proteins with
a high basic amino acid content.
Coomassie Stains
Coomassie (Brilliant) Blue is the most common
stain for protein detection in polyacrylamide gels.
Coomassie R-250 and G-250 are fabric dyes that have
been adapted to stain proteins in gels. The “R” and “G”
designations indicate red and green hues, respectively.
These stains generate visible protein patterns that can
be analyzed using densitometric methods.
Silver Stains
Silver stains offer high sensitivity but with a low
linear dynamic range (Merril et al. 1981). Often, these
protocols are time-consuming and complex. Silver
staining protocols have multiple steps with critical
timing; for this reason, they can be insufficiently
reproducible for quantitative analysis. In addition,
their compatibility with mass spectrometric protein
identification techniques is lower than Coomassie
stains and fluorescent dyes. There are many different
silver staining techniques with differing chemistries
and sensitivities.
Fluorescent Stains
Fluorescent stains fulfill almost all the requirements for
an ideal protein stain by offering high sensitivity, a wide
linear dynamic range (up to four orders of magnitude),
a simple and robust protocol, and compatibility with
mass spectrometry. These sensitive stains generate
little background and are easy to use.
Because fluorescent stains require specialized
instrumentation for imaging, the choice of stain
may be dictated by the instrumentation available.
Fluorescent dyes absorb light at one wavelength
and re-emit the light at another longer wavelength.
Imaging instruments differ in both the type of light
delivered for absorbance and the capabilities for
detecting the emitted light. The simplest and least
expensive systems use UV transillumination and a
camera for image capture; however, not all fluorescent
stains are optimally excited by UV light. Other imaging
systems use laser light to scan the gel. Laser light
is monochromatic, and the laser must be selected
according to the absorbance properties of the dye.
Not all fluorescent gel stains absorb visible light at
wavelengths supplied by imaging lasers.
Theory and Product Selection
Fluorescent stains can be at least as sensitive as
silver stains and are, therefore, subject to some of
the same potential problems stemming from high
sensitivity. Clean technique is essential, as any dust or
dirt transferred to the surface of the gel may appear
in the fluorescence image as smudges or speckles.
Contaminant proteins such as keratin will also appear
in the gel image if care is not taken to minimize such
contamination.
All fluorescent reagents are subject to photobleaching
to varying degrees. The fluorescent stains discussed in
the Protein Stains sidebar are reasonably photostable
and do not degrade noticeably through routine
exposure to room light during a staining procedure.
However, avoid exposure of the gel or staining solution
to intense light and cover the staining tray with an
opaque lid or foil.
Negative Stains
These rapid stains require only ~15 min for highsensitivity staining and generate protein bands that
appear as clear areas in a white background. Zinc and
copper stains do not require gel fixation and proteins
are thus not altered or denatured. Negative stains
can be used as a quality check before transferring
to a western blot or analysis by mass spectrometry,
though they are not the best choice when quantitative
information is desired.
Stain-Free™ Technology
This proprietary Bio-Rad technology allows protein
detection in a gel both before and after transfer,
as well as total protein detection on a blot when
using wet PVDF membranes, without the need for
application of a stain (see sidebar).
57
Chapter 5: Detection
2-D Electrophoresis Guide
Protein Stains
(contd.)
Coomassie Stains
Bio-Rad total protein stain selection guide.
Total Protein Stain
Detection Method
Coomassie Stains
Sensitivity
(Lower Limit) Time
Comments
Visible
MS
Compatible?
Coomassie (Brilliant) Blue 36–47 ng
2.5 hr
R-250
Simple and consistent; requires destaining with methanol
Yes
8–28 ng 1–2.5 hr
Bio-Safe™ Coomassie G-250
Nonhazardous staining in aqueous solution; premixed
Yes
Silver Stains
Visible
Silver stain 0.6–1.2 ng
2 hr
(Merril et al. 1981)
Stains glycoproteins, lipoproteins, lipopolysaccharides, nucleic acids
No
0.6–1.2 ng
1.5 hr
Silver Stain Plus™ kit
Simple, robust protocol (Gottlieb and Chavko 1987)
Limited
0.25–0.5 ng
3 hr
Dodeca™ silver stain kit
Simple, robust protocol; ideal for use with Dodeca stainers (Sinha et al. 2001)
Yes
Oriole fluorescent
0.5–1 ng
1.5 hr
gel stain
Rapid protocol requires no fixing or destaining; requires UV excitation
Yes
Flamingo fluorescent 0.25–0.5 ng
5 hr
gel stain
Simple protocol requires no destaining; high sensitivity, broad dynamic range;
excellent for laser-based scanners
Yes
Fluorescent Stains
Fluorescence
SYPRO Ruby protein 1–10 ng
Overnight Simple, robust protocol; gel stain
broad dynamic range
Negative Stains
Yes
Visible
Zinc stain
6–12 ng
15 min
High-contrast results; simple, fast, and
reversible; compatible with elution or blotting
as well (Fernandez-Patron et al. 1992)
Yes
Copper stain
6–12 ng
10 min
Single reagent; simple, fast protocol and reversible stain; compatible with elution or
blotting as well (Lee et al. 1987)
Yes
Stain-Free Technology
Stain-Free
8–28 ng
5 min
No separate staining steps
Yes, but fluorescencetryptophan
residues are modified
Coomassie Brilliant Blue R-250
Oriole Fluorescent
Gel Stain
58
Coomassie R-250 staining solution is prepared for
a traditional staining procedure in which gels are
stained in a methanol-water-acetic acid solution of
Coomassie R-250 dye. It requires ~40 ng protein
per spot for detection, though absolute sensitivity
and staining linearity depend on the proteins
being stained.
Bio-Safe Coomassie stain is a ready-to-use, singlereagent protein stain made with Coomassie (Brilliant)
Blue G-250. It offers sensitivity similar to colloidal
Coomassie stains (down to 8 ng) and a rapid
staining protocol. No additional reagents besides
water are required.
Fluorescent Stains
Theory and Product Selection
Silver Stain Kit
Flamingo Fluorescent
Gel Stain
Silver Stain Plus Kit
Zinc Stain and
Destain Kit
Flamingo fluorescent gel stain is prepared from
a dye that binds denatured protein. Normally
non-fluorescent in solution, it becomes strongly
fluorescent when bound to protein. There is,
therefore, no need for destaining, since unbound
dye in the gel is only minimally fluorescent.
A prolonged fixing step is necessary to wash
buffers and SDS out of the gel prior to staining,
as these substances can prevent dye binding.
Flamingo fluorescent gel stain is the most sensitive
of the listed fluorescent stains, with sensitivity to
0.25–0.5 ng, and it can be linear over three orders of
magnitude. The simple two-step staining procedure
can be completed in as little as five hours.
With a primary fluorescence excitation maximum at
512 nm and a considerably weaker excitation peak
at 271 nm, Flamingo fluorescent gel stain gives
the most sensitive results when imaged with laser
fluorescence scanning instruments equipped with
green or blue laser light sources. UV transilluminatorbased systems may also be used, but extended
exposure times may be required and sensitivity will
not be as high.
Oriole fluorescent gel stain is sensitive and, of the
stains listed, it is the easiest and most rapid to use.
The one-step staining process does not require
fixation or destaining, allowing protein samples
to be accurately visualized and quantitated in less
than two hours. Since SDS is required for optimal
staining, prior fixing or washing of the gel can impair
staining sensitivity.
The dye in Oriole stain is excited only weakly by
wavelengths longer than 400 nm and can, therefore,
only be imaged using UV-based imaging systems.
Oriole’s limit of detection is 1 ng or less in a typical
protein spot.
SYPRO Ruby was one of the original fluorescent
protein gel stains, and it has a combination of high
sensitivity and wide dynamic range that cannot be
achieved with traditional Coomassie blue or silver
stains. SYPRO Ruby has two prominent absorbance
peaks, one at ~270 nm in the UV range and the
other at ~460 nm in the visible range. This allows
imaging with both UV transilluminator and laserscanning systems. Detection sensitivity in SYPRO
Ruby–stained gels can be as low as 1 ng. SYPRO
Ruby stains most classes of proteins with little
protein-to-protein variability.
The principle advantage of SYPRO Ruby is its
versatility with respect to imaging requirements.
It is, however, time-consuming to use and does
not produce the high-quality mass spectrometric
data generated with other fluorescent stains
(Berkelman et al. 2009).
Copper Stain
59
Chapter 5: Detection
2-D Electrophoresis Guide
Dodeca High-Throughput Stainers
(contd.)
Silver Stains
Three silver staining methods are recommended
for use with 2-D gels. Though they are based on
slightly different chemistries, they have similar
protein sensitivities.
The Bio-Rad silver stain kit, based on the method
of Merril et al. (1981), can be up to 100 times more
sensitive than Coomassie Blue R-250 dye staining
and allows visualization of heavily glycosylated
proteins in gels. Protein spots containing 10–100 ng
of protein can be easily seen. Proteins in gels are
fixed with alcohol and acetic acid, then oxidized
in a solution of potassium dichromate in dilute nitric
acid, washed with water, and treated with silver
nitrate solution. Silver ions bind to the oxidized
proteins and are subsequently reduced to metallic
silver by treatment with alkaline formaldehyde.
Color development is stopped with acetic acid
when the desired staining intensity has been
achieved. This method is not compatible with mass
spectroscopic analysis since the oxidative step
affects protein mass.
Silver Stain Plus stain from Bio-Rad, based on
the method developed by Gottlieb and Chavko
(1987), requires only one simultaneous staining and
development step. Proteins are fixed with a solution
containing methanol, acetic acid, and glycerol and
then washed extensively with water. The gels are
then soaked in a solution containing a silver-amine
complex bound to colloidal tungstosilicic acid.
Silver ions transfer from the tungstosilicic acid to the
proteins in the gel by means of an ion exchange or
electrophilic process. Formaldehyde in the alkaline
solution reduces the silver ions to metallic silver to
produce the images of protein spots. The reaction is
stopped with acetic acid when the desired intensity
60
Theory and Product Selection
has been achieved. Silver ions do not accumulate
within the gel, so background staining is light.
Since this method lacks an oxidizing step,
visualization of heavily glycosylated proteins and
lipoproteins can be less sensitive than with the
Merril stain.
Dodeca silver stain is based on the method
described by Sinha et al. (2001), in which proteinbound silver ions are chemically reduced to form
visible metallic silver. This stain was developed for
use with the high-throughput Dodeca stainers and
can be used with mass spectrometry.
Dodeca stainers are high-throughput gel staining
devices available in two sizes: the small size
accommodates up to 24 Criterion gels while the
large size can accommodate up to 12 large-format
gels. The stainers feature a shaking rack designed
to hold staining trays at an angle to allow air bubbles
to escape and ensure uniform gel staining to protect
gels from breaking. Use of the stainers ensures
high-quality, consistent results and eliminates
gel breakage from excess handling. They are
compatible with the following stains:
■■
■■
Stain-Free Technology
■■
A special additive in Bio-Rad’s Criterion Stain Free™,
Criterion™ TGX Stain-Free™, and Mini-PROTEAN®
TGX Stain-Free™ gels covalently modifies tryptophan
residues when activated with UV light. This
enhances the proteins’ intrinsic fluorescence and
shifts the emission into the visible range (>400 nm),
allowing protein detection (with a stain-free
compatible imager, such as the Gel Doc™ EZ or
ChemiDoc™ MP systems) in a gel both before and
after transfer, as well as total protein detection on a
blot when using wet PVDF membranes.
Detection of Proteins on Western Blots
This system is ideal for quick sample assessment
during purification procedures and as a precursor
to blotting and profiling workflows in which
Coomassie (Brilliant) Blue staining is ordinarily
used. The sensitivity of the Stain-Free system is
comparable to that of staining with Coomassie
Blue for proteins with a tryptophan content >1.5%;
sensitivity superior to Coomassie staining is possible
for proteins with a tryptophan content >3%.
Proteins that do not contain tryptophan residues
are not detected.
2-D electrophoresis can be combined with
western blotting for monitoring the posttranslational
modification of trace proteins in complex mixtures and
evaluating the specificity of antibodies and antisera.
Numerous techniques are available for the transfer of
proteins to membranes and for the probing of western
blots with antibodies, stains, and other reagents.
These techniques are described in more detail in
the Protein Blotting Guide (bulletin 2895).
■■
■■
■■
High-Throughput Dodeca Stainers
Bio-Safe Coomassie (Brilliant) Blue G-250 stain
Coomassie (Brilliant) Blue R-250 stain
SYPRO Ruby protein gel stain
Flamingo fluorescent protein gel stain
Oriole fluorescent gel stain
Dodeca silver stain kits
Certain synthetic membranes bind proteins tightly
and can be used as supports for solid-phase
immunoassays, staining, or other analysis.
These membranes, known as western blots,
are useful for the identification of specific proteins
and protein modifications.
61
2-D Electrophoresis Guide
Theory and Product Selection
CHAPTER 6
Image Acquisition,
Analysis, and Spot Cutting
62
63
Chapter 6: Image Acquisition, Analysis, and Spot Cutting
2-D Electrophoresis Guide
Finding Protein Spots of Interest
■■
After 2-D gels are stained, the protein patterns can
be digitized and analyzed with an image evaluation
system comprising an imaging device and analysis
software. Following analysis, spots of interest can
be excised from gels for further analysis, by mass
spectrometry for example (see Chapter 7).
Image Acquisition
In proteomic applications, selecting the image
acquisition device depends on the staining technique
used. A number of imaging systems are capable of
multiple detection modes and can be used with a
variety of applications.
■■
ensitometers enable the visualization of gels
D
stained with visible light–absorbing stains such as
Coomassie, negative, or silver stains
■■
harge-coupled device (CCD) camera systems can
C
feature different light sources for greater application
flexibility. They can be used for visualization
of visible and fluorescent stains and of
chemiluminescence in some cases. Systems
offer transillumination (visible or UV light source
underneath the gel or blot) or epi-illumination
(colored or white light positioned above the sample).
Heat in the camera system can manifest as noise,
and this noise can prevent detection of faint
chemiluminescent signals above the background.
Supercooled CCD cameras reduce image noise,
allowing detection of faint signals
aser-based scanners offer the highest sensitivity,
L
resolution, and linear dynamic range. They are
powerful image acquisition tools for electrophoresis
gels and blots stained with fluorescent dyes.
Lasers can be matched to the excitation wavelengths
of a multitude of fluorophores
Theory and Product Selection
Imaging Systems
System Type and Application
GS-900™
Gel Doc™ EZ
DensitometerChemiDoc™ MP
PharosFX™ and
PharosFX Plus
Type of imager
Laser-based
Densitometer
CCD camera-based
CCD camera-based
Light source options
Epi- and Transillumination of UV
Transillumination of UV
transillumination of and white light*
and white light
white light
Epi-illumination by LEDs
(red, green, blue, and white)
488 nm external laser
532 nm internal laser
635 nm external laser
Optimized applications
Visible stains
•
•
•
—
UV light–excited
fluorescent stains
—
•
•
—
Visible light–excited
fluorescent stains and labels
—
•
—
•
Fluorescent multiplexing
—
•
—
•
Chemiluminescence
Stain-Free™
—
—
•
•
—
•
—
—
* White light conversion screen is required.
Bio-Rad’s GS-900 calibrated imaging densitometer
has transmittance and true reflectance capabilities
that allow accurate scans of samples that are
either transparent (gels and film) or opaque (blots).
It provides high-quality imaging to resolve close
spots and a variable resolution feature to preview
and crop images.
Bio-Rad’s ChemiDoc MP supercooled CCD
system provides maximum flexibility. It offers
transillumination of both UV light (for imaging UV
fluorescent stains) and white light (for imaging visible
stains). It also offers optional LED epi-illumination
in red, green, and blue for single fluorescent stains
or fluorescent multiplexing. In addition, it can also
image stain-free gels, which require no staining or
destaining and are ready for imaging in a matter of
minutes after completing the SDS-PAGE run.
Image Analysis
Following image acquisition, a robust software
package is required to analyze and present the
data and to draw conclusions from 2-D gel images.
The software should provide a variety of tools to
enhance the user’s ability to evaluate the acquired
data. For example, the software should be able to
adjust contrast and brightness and magnify, rotate,
resize, and crop gel images. It should measure total
and average quantities and determine relative amounts
of protein. It should also be capable of determining
64
The PharosFX systems use multiple lasers, which
enhance application flexibility and allow optimum
excitation of single- or multicolor fluorescent
samples to enable detection of most fluorescent
dyes and labels. Computer-controlled, useraccessible filter wheels have eight filter slots,
supporting multiplex or multicolor fluorescence
imaging applications in gels and blots, such as
Qdot multiplex blotting, DIGE, and gel staining with
Pro-Q stains. The Molecular Imager ® PharosFX
system has all the features of the PharosFX Plus
imager for fluorescence and visible detection,
but it lacks the storage phosphor option for
imaging radioisotopes.
the presence/absence and up- or downregulation of
proteins, their molecular weight, pI, and other values.
Following this initial analysis, computer-assisted
image analysis software should allow:
■■
■■
torage and structuring of large amounts
S
of collected experimental image data
apid and sophisticated analysis of
R
experimental information
■■
Supplementation and distribution of data among labs
■■
Establishment of 2-D-protein data banks
65
Chapter 6: Image Acquisition, Analysis, and Spot Cutting
2-D Electrophoresis Guide
Data Analysis and Reporting
PDQuest™ 2-D Analysis Software
Bio-Rad’s PDQuest 2-D analysis software is used
for analyzing and creating databases for 2-D
electrophoresis gels. It provides a series of “wizards”
for the analysis of digitized gel images and for spot
detection and quantitation, gel comparison, and
statistical analysis. The Experiment Wizard guides
selection of gels for analysis, detection of spots
of interest, creation of an experiment, and matching
of gels. The Spot Detection Wizard then guides the
identification and quantitation of the
spots in gel images.
After detection, gels in the same series are placed
in an experiment for comparison, statistical
analysis, and databasing. Histograms allow quick
comparisons of the quantities of the same spot
in all the gels in an experiment. Spots can also
be compared qualitatively, organized into userdefined sets for further analysis, and annotated
and databased for easy identification. Spots from
different experimental series can be organized and
compared in higher-level experiments. PDQuest can
be used to simultaneously analyze thousands of
spots on hundreds of gels. Data can be exported
to other applications, such as spreadsheets, for
further analysis.
PDQuest software has the further advantage of
integration with Bio-Rad’s EXQuest™ spot cutter,
which accurately locates and excises protein
spots from 2-D gels or blots at high speed
(up to 600 spots per hour) and then loads them
into 96- or 384-well microplates or 96-tube
racks for downstream processing and analysis.
PDQuest has no imaging functions besides driving
the camera system in the ExQuest spot cutter, but it
can read and import multiple file formats from other
gel imaging software packages like Quantity One®.
With PDQuest software, all gels in a single experiment
are viewed as a unit. To compare gels from different
experiments, the reference images are compared.
In such comparisons, each spot is automatically
assigned a number such that identical spots have
identical numbers. In an experiment, the molecular
weight and pI values for known protein spots can
also be entered. With these data, PDQuest can
estimate molecular weight and pI values for all the
spots in the experiment.
Analysis sets allow the study of sets of proteins that
are statistically and scientifically significant and to
identify spots to cut using the ExQuest spot cutter.
There are six different kinds of analysis sets:
■■
■■
■■
Image Optimization, Spot Detection, and Quantitation
Before any software can detect the protein spots of a
2-D gel, raw image data must be optimized and the
gel background subtracted.
PDQuest software models protein spots
mathematically as 3-D Gaussian distributions and
uses the models to determine protein maxima.
A 3-D Gaussian spot is a precise representation of an
original scanned spot. Gaussian curves are fitted to
the scanned spot in the X and Y dimensions, and then
additional modeling is performed to create the final
Gaussian spot. Using Gaussian modeling, it is possible
to accurately quantitate overlapping spots, spots in
gel streaks, and multiple spots in dense clusters.
The accuracy of automatic spot detection depends
on the quality of the 2-D gels and their images.
Correction capabilities of PDQuest software can be
used to add undetected spots to the list of spots or
to delete spots that arise from gel artifacts.
Gel Comparison
The next step in 2-D gel evaluation is identification
of proteins that are present in all gels of a series.
Since inherent problems with gel-to-gel reproducibility
affect the positions of spots within a gel series,
gel analysis software must be able to detect minor
shifts in individual spot position within the gel series.
66
Many software packages for automatic gel comparison
are created with the assumption that the relative
positions of spots are altered only slightly relative to
each other, and they allocate the spots on this basis.
Prior to automatic gel comparison, PDQuest software
selects the best 2-D gel of a gel series as a reference,
or standard gel, and compares all other 2-D gels to
this gel. Proteins in a gel series that are not present in
the reference gel can be added automatically so that
the reference gel includes all proteins of a gel series.
PDQuest includes the ability to match spots with no
manual assistance, and it is possible to display up
to 100 enlarged details of 2-D gels on the screen
simultaneously, enabling rapid and error-free
determination of the matching quality.
Data Normalization
When comparing gels in an experiment, there is
often some variation in spot size and intensity among
gels that is not due to differential protein expression.
Multiple normalization methods can be used to
compensate for gel-to-gel variations in spot intensity
caused by inconsistencies in sample loading, gel
staining, and imaging. To accurately compare spot
quantities among gels, compensation for these
variations in spot intensity, which are not related to
expression levels, is required.
■■
■■
■■
ualitative analysis sets — spots that are present
Q
in one gel but not in another
uantitative analysis sets — spots whose intensity
Q
(amount) has increased or decreased by a certain
degree, or whose intensity has changed above,
below, or within the fold change factor that you specify
tatistical analysis sets — spots that are significant
S
according to the statistical test that you apply
Theory and Product Selection
EXQuest Spot Cutter
Bio-Rad’s EXQuest spot cutter accurately locates
and excises protein bands or spots from gels
or blots and loads them into 96- or 384-well
microplates or 96-tube racks for downstream
processing and analysis. Its camera works with
PDQuest 2-D analysis software to visualize gels
and blots that are either visibly or fluorescently
stained. In 2-D electrophoresis applications,
PDQuest software tracks the protein bands
or spots through spot cutting and protein
identification, which is usually accomplished
using mass spectrometry.
The EXQuest spot cutter allows use of any common
proteome separation and staining methods:
■■
■■
■■
reestanding or plastic- or glass-backed 2-D
F
and 1-D SDS-PAGE gels
PVDF and nitrocellulose membrane blots
els or membranes stained for proteins with
G
visible stains (such as silver and Coomassie blue
stains) or fluorescent stains (such as Flamingo™,
Oriole™, and SYPRO Ruby protein stains)
Arbitrary analysis sets — manually selected spots
oolean analysis sets — created by comparing
B
two or more analysis sets (for example, set C could
be made up of those spots present in both sets
A and B)
atching analysis sets — spots that are either
M
unique to one member or present in all members
Once proteins of interest are determined, the
corresponding analysis sets are uploaded to the spot
cutter. The spots of interest are then excised from the
gels and digested to release peptides for analysis by
various mass spectrometry methods.
Spot Cutting from 2-D Gels
Spots of interest can be excised from gels either
manually (for example, with a scalpel, razor blade,
or modified pipet tip) or with an automated spot
cutting system. The advantages of automated systems
are numerous and include improved precision and
reproducibility, tracking of gel spots, and decreased
risk of contamination. The excised gel plugs are
then transferred to microplates or other vessels for
digestion and further analysis.
ExQuest Spot Cutter
67
2-D Electrophoresis Guide
Theory and Product Selection
CHAPTER 7
Identification and
Characterization of
2-D Protein Spots
68
69
Chapter 7: Identification and Characterization of 2-D Protein Spots
2-D Electrophoresis Guide
Beyond Excision
2-D electrophoresis has the unique capability of
simultaneously displaying several hundred proteins.
When coupled with the ability of mass spectrometry
to identify and characterize small quantities of protein,
2-D electrophoresis is a very powerful and effective
analytical method.
Several mass spectrometric techniques can be
used for protein identification at the end of a 2-D
electrophoresis workflow. Most of these methods first
require proteolytic digestion of the protein into discrete
fragments that can be eluted from the excised gel
plug. The most basic mass spectrometric method,
peptide mass fingerprinting, simply determines
accurate masses of the peptides generated.
These masses are then compared to a database,
and the protein of origin can often be uniquely
identified. Another technique, tandem mass
spectrometry (MS/MS) further fragments selected
peptides along the peptide backbone, allowing the
generation of limited sequence information that can
be used to refine the protein identification step.
In-Gel Proteolytic Digestion
Proteolytic digestion can be performed directly on
processed gel pieces. Because proteases are also
subject to autolysis, always include a blank piece as a
control. Proteases used for this purpose are selected
for their efficiency in in-gel digestion and for their
defined cleavage specificity, which allows prediction of
the generated peptide masses. The most commonly
used protease is trypsin, but other proteases used
include LysC, GluC, ArgC, AspN, and LysN, which
cleave to either the C- or N-terminal side of a single
amino acid, as signified by their nomenclature.
These enzymes are all commercially available as
preparations that have been specifically modified for
use prior to mass spectrometry. Enzymes specifically
recommended for mass spectrometry should always
be used for in-gel digestion.
■■
■■
Proteolytic Digestion
In-gel digestion (Rosenfeld 1992) of selected proteins
is part of the sample preparation process for mass
spectrometry, and it comprises four basic steps:
destaining (washing) the gel pieces, reduction and
alkylation, proteolytic cleavage of the protein,
and extraction of the resultant peptides.
Washing
After excision of the protein spot of interest from the
gel, most protocols require destaining of the proteins
before proceeding. The destaining or wash protocol
depends on the stain used for visualization.
Commonly used protocols for various stains are
described in Part II of this guide.
Reduction and Alkylation
Reduction and alkylation together reduce and
irreversibly block the formation of inter- and
intramolecular disulfide bridges, which can significantly
improve the efficacy of proteolytic cleavage and
subsequent mass spectrometry.
Proteins excised from 2-D gels have usually been
reduced and alkylated either during sample
preparation or equilibration prior to the second
dimension and may not require this step. This step
is mandatory if upstream processing did not
incorporate reduction and alkylation. Any reduction
or reduction plus alkylation step must be followed
by a cleanup step prior to mass spectrometry.
70
■■
■■
se trypsin (modified porcine pancreatic trypsin,
U
mass spectrometry grade) for initial protein digestion.
Trypsin is one of the most specific proteases and
cleaves at the C-terminal side of Arg and Lys
se GluC, AspN, or LysC with proteins of smaller
U
mass. These enzymes generate fewer peptides
of larger mass than trypsin, which may generate
fragments too small for definitive identification
se acid hydrolysis, cyanogen bromide cleavage,
U
or other chemical methods if alternatives to
enzymatic digestion are required
ome proteins are processed forms of larger
S
proteins; therefore, once the protein is identified
based on a trypsin digestion, other methods can be
used to define the N- and C-termini of the fragment
he resulting peptides can be extracted with
T
acetonitrile, dried under vacuum, and dissolved in a
small amount of water. Prior to mass spectrometry,
the samples should be further purified by solid phase
extraction, for example using ZipTip pipet tips.
A protocol is provided in Part II of this guide.
Identification by Mass Spectrometry
– ESI — a flowing liquid is passed through a
charged orifice to produce charged droplets,
which are then desolvated to yield gas-phase
peptide ions. ESI can be coupled directly to liquidphase separations such as chromatography
(LC-MS) and generates multiply-charged
molecular ions that bring mass-to-charge ratio
(m/z) values within the mass range of mass
spectrometry instruments most commonly
used with ESI
Identification of the peptides derived from digestion
can be achieved using several mass spectrometry
techniques. Only a brief overview of mass
spectrometry theory and techniques is presented here.
Refer to the literature from mass spectrometer vendors
for more information about systems and methods.
Mass spectrometry systems contain the following
components (Figure 7.1):
■■
Ionization source — converts the sample into
gas-phase ions, which are then injected into a mass
analyzer. The two ionization sources most commonly
used for peptide mass spectrometry are matrixassisted laser desorption ionization (MALDI) and
electrospray ionization (ESI)
–MALDI — the protein is mixed with an organic
molecule (the “matrix”), deposited onto a planar
substrate, allowed to dry, and illuminated with a
pulsed UV laser. The matrix compound absorbs
the laser energy and promotes peptide ionization,
typically generating singly-charged molecular ions.
MALDI is useful for high-throughput applications
but is limited by ion suppression (particularly in
complex peptide mixtures) and chemical noise
from the matrix in the low mass range
Theory and Product Selection
■■
■■
Mass analyzer — sorts the ions according the m/z.
A number of different types of mass analyzers are
available, including time-of-flight (TOF), quadrupole,
and ion trap systems as well as combinations of
these (hydrid mass spectrometers)
Ion detector — records the ion current, amplifies it,
and sends it to the data analysis system where it is
presented in the form of a mass spectrum. The m/z
values of the ions are plotted against their intensities
to show the number of components in the sample,
the molecular mass of each component, and the
relative abundance of the various components in
the sample
The data from the mass analyzer(s) are used for protein
identification, and two options are most common
in the 2-D electrophoresis workflow: peptide mass
fingerprinting and tandem mass spectrometry.
Sample
Introduction
Ionization
Source
Mass
Analyzer
Detector
Data
Handling
Fig. 7.1. Components of a mass spectrometer.
71
Chapter 7: Identification and Characterization of 2-D Protein Spots
2-D Electrophoresis Guide
of peptide mass fingerprinting, however, include the
following: (i) the protein sequence has to be present
in the database of interest, and (ii) several peptides
are required to uniquely identify a protein. Additionally,
most algorithms assume that the peptides come from
a single protein, which is why resolution in the 2-D
separation is so critical. If this information does not
allow unequivocal identification of the protein, peptides
can then be analyzed by tandem mass spectrometry.
Peptide Mass Fingerprinting
In this method, the peptides resulting from digestion
of the protein of interest are analyzed by mass
spectrometry and compared to a database of
calculated peptide masses generated by “in silico”
cleavage of protein sequences using the same
specificity as the enzyme that was employed in the
experiment. Identifications (“hits”) are scored in terms
of confidence of match (Figure 7.2).
Tandem Mass Spectrometry (MS/MS)
In MS/MS, a peptide ion is isolated in the mass
analyzer and subjected to dissociation to product ion
fragments. Peptides dissociate according to certain
rules. For example, fragmentation typically occurs
along the peptide backbone; each residue of the
peptide chain is successively cut off, both in the N->C
(a-, b-, c- ions) and C->N (x-, y-, z- ions) directions.
The product ions resulting from the fragmentation
are analyzed in a second stage of mass analysis,
which enables sequence derivation (Figure 7.3).
Tandem MS can allow identification of proteins
from a single peptide (Lovric 2011).
This approach requires simple mixtures of proteins or
pure proteins and is, therefore, suitable for analysis of
proteins isolated from 2-D electrophoresis. Limitations
Theory and Product Selection
Establishment of 2-D Databases
After the spots are cut, analyzed, and identified, by
MS for example, the information can be imported
back into the experiment as annotations. Annotations
are organized in categories, for example by protein
name, protein family amino acid composition,
protein function, cellular location, binding properties,
and translational regulation. A single spot may be
annotated in multiple categories, depending on the
amount and type of information available about it.
Most categories contain simple text annotations.
Specialized categories can be used to link spots
to Internet protein databases or to open files in
other applications.
N
C
N
C
ENIYPEDQQESPSIGLK
N
N
NQNEYQVSWDTEK
Ion current
C
AWGISPVR
N
N
VQVSR
C
C
QGLWIVDMSSGAVK
C
Digestion
MS
m/z
WGISPVR
AWGISPV
AWGISP
ISPVR
AWGIS
SPVR
AWGI
Ion current
Abundance
Analysis
MS/MS
m/z
Fig. 7.2. Peptide mass fingerprinting. Peptides resulting from digestion are analyzed by mass spectrometry, and the resulting m/z values
and mass spectrum are compared to theoretical values derived from “in silico” digestion of known proteins in a database.
72
m/z
Fig. 7.3. MS/MS analysis. The first mass analyzer selects ions of a particular m/z for fragmentation. The second mass analyzer produces the
mass spectrum for those fragments.
73
2-D Electrophoresis Guide
Methods
TABLE OF CONTENTS
PART II
Methods
CHAPTER 8
Sample Preparation
74
75
2-D Electrophoresis Guide
Chapter 8: Sample Preparation
Buffers and Solutions
Tips for Sample Preparation
Keep the sample preparation workflow simple
(increasing the number of sample handling steps
may increase variability).
■■
Lysis (Cell Disruption)
■■
■■
or each 10 mg (fresh weight) pelleted cells or
F
animal tissue, use about 1 ml of 2-D sample
solution for a protein concentration of 1–3 mg/ml.
When disrupted in liquid nitrogen, samples such
as liver biopsies and plant leaves contain 10–30%
and 1–2% extractable protein, respectively
■■
—Disrupt the sample or place freshly disrupted
samples in solutions containing strong
denaturing agents such as 7–9 M urea,
2 M thiourea, or 2% SDS. In this environment,
enzymatic activity is often negligible
—Perform cell disruption at low temperatures
to diminish enzymatic activity
—Add a chemical protease inhibitor
to the lysis buffer. Examples include
phenylmethylsulfonyl fluoride (PMSF),
aminoethyl-benzene sulfonyl fluoride (AEBSF),
tosyl lysine chloromethylketone (TLCK),
tosyl phenyl chloromethyletone (TPCK),
ethylenediaminetetraacetic acid (EDTA),
benzamidine, and peptide protease inhibitors
(for example, leupeptin, pepstatin, aprotinin,
bestatin). For best results, use a combination
of inhibitors in a protease inhibitor cocktail
—If protein phosphorylation is to be studied,
include phosphatase inhibitors such as fluoride
and vanadate
■■
■■
■■
76
hen working with a new sample, use at least two
W
different cell disruption protocols and compare
the protein yield (by protein assay) and qualitative
protein content (by SDS-PAGE)
ptimize the power settings of mechanical
O
rupture systems and incubation times for all
lysis approaches. Mechanical cell lysis usually
generates heat, so employ cooling where required
to avoid overheating of the sample
irect application of clarified lysate to IPG strips
D
is appropriate only for samples with high protein
content and minimal interfering substances.
Preparation of many sample types (for example,
plant tissues and dilute bodily fluids) should
incorporate a precipitation step to remove
interfering substances and allow application
of a more concentrated sample
Protein Solubilization
To diminish endogenous enzymatic activity:
—Lyse samples at pH >9 using either sodium
carbonate or Tris as a base in the lysis solution
(proteases are often least active at basic pH)
Methods
■■
■■
■■
■■
■■
■■
repare fresh sample solubilization solutions
P
daily or store them frozen in aliquots, preferably
at –80°C; always use high-quality reagents and
proteomics-grade water. Use urea stock solutions
soon after they are made, or treat them with a
mixed-bed ion exchange resin to avoid protein
carbamylation by cyanate, which forms in old urea.
If solutions are prepared in advance and stored,
it is best to prepare them without reductant (DTT)
and add the reductant directly before use
issolve pelleted protein samples in 1×
D
2-D sample solution
erform a protein quantitation assay to determine
P
the amount of total protein in each sample. Use a
protein assay that is tolerant to chemicals in your
samples. For samples in 2-D sample solution,
for example, use the RC DC™ protein assay,
which can tolerate up to 2% detergent
ilute or concentrate samples as needed to
D
yield a final protein concentration of 1–5 mg/ml.
Make dilutions in 2-D sample solution and
concentrate the sample using the ReadyPrep™
2-D cleanup kit
se protein extracts immediately or aliquot them
U
into appropriately sized batches and store them
at –80°C to avoid freeze-thaw cycles
ighly viscous samples likely have a very high
H
DNA or carbohydrate content. Fragment DNA
with ultrasound during protein solubilization or by
adding endonucleases like benzonase. Use protein
precipitation (for example, with the ReadyPrep 2-D
cleanup kit) to diminish carbohydrate content
2-D sample solution (50 ml) Cell washing buffer (1 L)
7 M urea, 2 M thiourea, 4% (w/v) CHAPS, 40 mM DTT,
0.2% (w/v) ampholytes (pH 3–10)
10 mM Tris-HCl, pH 7.0, 250 mM sucrose
Urea/thiourea stock solution
CHAPS
Bio-Lyte® ampholytes, pH 3–10
DTT
Bromophenol blue (1%)
Distilled or deionized H2O ■■
■■
■■
48 ml
2.0 g
250 µl
0.31 g
10 µl
to 50 ml
-D sample solution is used for sample application
2
and IPG strip rehydration. Bio-Rad offers various
types of 2-D sample buffers, which differ in
solubilizing power (see Ordering Information)
or pH control, Tris base may be added to the
F
2-D sample solution at 10–40 mM. Addition of
Tris increases the conductivity of the sample solution
and extends the time required to focus the IPG strips
mpholytes are added to all IPG rehydration
A
and sample solubilization solutions to maintain
solubility of the proteins. The choice of ampholytes
depends on the pH range of the IPG strip.
Higher concentrations (up to 1% (w/v)) may be
used, but they result in lower IEF voltage and
correspondingly longer focusing times
Urea/thiourea stock solution (50 ml)
Urea
22 g
Thiourea
8g
Distilled or deionized H2O
to 50 ml
Filter through Whatman No. 1 paper
using a Buchner funnel
Store at –80°C
1% Bromophenol blue (10 ml)
Bromophenol blue will not dissolve in unbuffered
water. Prepare 10 ml of 50 mM Tris base by dissolving
60.6 mg of Tris in 10 ml of water. Add 100 mg of
bromophenol blue and vortex until dissolved.
Store at 25°C.
Tris base
Sucrose
Distilled or deionized H2O
1.21 g
85.58 g
800 ml
Dissolve
Adjust pH to 7.0 with HCl
Distilled or deionized H2O
Store at 4°C
to 1 L
Protein precipitation solution (100 ml)
20% (w/v) trichloroacetic acid (TCA), 0.2% DTT (w/v)
in ice-cold acetone (–20°C)
Trichloroacetic acid
DTT
Acetone
Dissolve
Acetone Store at –20°C
20 g
0.2 g 80 ml
to 100 ml
Wash solution (100 ml)
0.2% DTT in ice-cold acetone (–20°C)
DTT Acetone
Dissolve
Acetone Store at –20°C
0.2 g 80 ml
to 100 ml
SDS sample solubilization buffer (50 ml)
1% (w/v) SDS, 100 mM Tris-HCl (pH 9.5)
SDS Tris base Distilled or deionized H2O
Titrate to pH 9.5 with diluted HCl
Distilled or deionized H2O
Store at 25°C
0.5 g
0.6 g
40 ml
to 50 ml
o not heat samples containing urea and thiourea
D
above 35°C as this can lead to protein modification
ollowing cell disruption, check the efficacy of
F
cell wall disruption by light microscopy and
centrifuge all extracts extensively (20,000 × g
for 15 min at 15°C) to remove any insoluble
material; solid particles may block the pores
of the electrophoresis gel
77
2-D Electrophoresis Guide
Chapter 8: Sample Preparation
Methods
Cell Lysis and Protein Extraction Procedures
Suspension Cultured Human Cells
Monolayer Cultured Human Cells
Mammalian Tissue
Microbial Cultures
Use the MicroRotofor™ cell lysis kit (mammalian) or the
protocol below, which uses 2-D sample solution and a
sonicator for cell lysis and protein extraction. Use 0.5 ml
of 2-D sample solution with 3 × 107 cells.
Use the MicroRotofor cell lysis kit (mammalian) or
the protocol below, which uses 2-D sample solution
and a sonicator for cell lysis and protein extraction.
Use 0.5 ml of 2-D sample solution with 3 × 107 cells.
Reagents
Reagents
Use the MicroRotofor cell lysis kit (mammalian) or the
protocol below, which involves freezing tissue samples
(for example, biopsy samples) in liquid nitrogen.
Use liquid nitrogen and a mortar and pestle to grind
the samples while they are still frozen. Break up any
larger pieces beforehand (for example, wrap the
frozen tissue sample in aluminum foil and crush
with a hammer).
Reproducible sample preparation from bacteria and
yeast is challenging because the cells may release
proteases and other enzymes into the growth medium
(Harder et al. 1999, Drews et al. 2004, Poetsch and
Wolters 2008). Wash the cultures thoroughly with isotonic
buffers and take precautions to inactivate the proteolytic
activity after cell lysis. Extensive disruption of microbial
cells is required and is usually performed with the help of
a French press, bead impact instruments, or sonicator.
■■
■■
2-D sample solution
Cell washing buffer
Protocol
ellet the cells by centrifugation at
P
2,000 × g for 5 min at 4°C.
2
Discard the supernatant and wash
pelleted cells in cold cell washing buffer.
Repeat steps 1 and 2 two times.
3
Add 2-D sample solution to the
pelleted cells and suspend the pellet
with a pipet.
5
6
■■
2-D sample solution
Cell washing buffer
Protocol
1
4
■■
Place the cell suspension on ice,
incubate 5 min, and sonicate at
appropriate intervals. Check lysis
efficacy by light microscopy.
Centrifuge cell debris at 14,000 × g
for 15 min and transfer supernatant
to a new vial.
erform a protein assay of the
P
supernatant. A protein concentration
of 3–5 mg/ml is best for
2-D electrophoresis.
1
2
3
4
5
6
Reagents
arefully remove (decant) culture
C
medium from cells. Wash cells twice
with cell washing buffer.
Add 2-D sample solution to the cells
and keep on ice for 5 min. Swirl the
plate occasionally to spread the buffer
around the plate.
Use a cell scraper to collect the lysate
and transfer to a microcentrifuge tube.
Place the cell suspension on ice,
incubate 5 min, and sonicate at
appropriate intervals. Check lysis
efficacy by light microscopy.
Centrifuge the cell debris at 14,000 × g
for 15 min and transfer the supernatant
to a new vial.
Perform a protein assay of the
supernatant. A protein concentration
of 3–5 mg/ml is best for
2-D electrophoresis.
■■
2-D sample solution
Protocol
1
2
3
Immediately after grinding, transfer
60 mg tissue powder to a
microcentrifuge tube containing
1.0 ml of 2-D sample solution.
Optional: sonicate the sample on
ice 5 times, for 2 sec each time.
Pause between sonication steps
to avoid overheating.
Reagents
■■
■■
■■
SDS sample solubilization buffer
2-D sample solution
Cell washing buffer
Protocol
1
Centrifuge cells (~5 × 107) at 5,000 × g
for 3 min and resuspend the pellet in an
equal volume of 2-D cell washing buffer
heated at 37°C and centrifuge again.
Repeat two more times to remove
all interfering material (extracellular
proteases and growth media).
4
Incubate the sample at room
temperature for 30 min. Vortex from
time to time.
5
Centrifuge at 35,000 × g for 30 min
at room temperature.
2
Perform a protein assay to determine
the protein concentration of the
supernatant, which should be
5–10 mg/ml.
Add ~150 µl hot (95°C) SDS sample
solubilization buffer to the pellet and
vortex thoroughly.
3
Sonicate the sample solution 10 times
for 1 sec each at ~60 W and ~20 kHz.
6
7
78
Chill a mortar with liquid nitrogen,
then grind small tissue pieces in
the presence of liquid nitrogen to
a fine powder.
Use the MicroRotofor cell lysis kit (bacteria),
the MicroRotofor cell lysis kit (yeast), or the protocol
below. This protocol relies on cell lysis with ultrasonic
waves in combination with a solubilization in SDS
under elevated temperature to ensure deactivation
and denaturation of proteases.
ilute the supernatant with 2-D
D
sample solution and incubate for
20 min at room temperature.
4
5
Incubate the sample at 95°C for 5 min.
Cool the sample to 20°C and dilute
with ~500 µl of 2-D sample solution.
Incubate for another 20 min at room
temperature. The final SDS concentration
should not exceed 0.25% in the extract
to be applied onto the IPG strip; therefore,
be sure that the total volume is
maintained during the SDS boiling step.
6
Centrifuge the sample solution at 20°C
for 30 min at 14,000 × g and harvest
the supernatant.
7
erform the protein assay. The protein
P
concentration should be ~5 µg/µl.
79
2-D Electrophoresis Guide
Chapter 8: Sample Preparation
Cell Lysis and Protein Extraction Procedures (contd.)
Sample Cleanup
Plant Leaves
Prior to IEF, remove contaminating salts, buffers, and other chemicals from samples by dialysis, precipitation,
or buffer exchange. A protocol for buffer exchange using Bio-Rad’s Micro Bio-Spin™ P-6 columns is provided
here. Another alternative is the use of the ReadyPrep 2-D cleanup kit to effectively precipitate sample protein
and remove contaminants. It has the additional benefit of concentrating the sample to a desired volume.
Plant leaf cells contain reactive compounds (such as proteases, phenol oxidases, organic acids, phenols,
and terpenes). To minimize the deleterious effects of these compounds on protein integrity, use the MicroRotofor
cell lysis kit (plant) or follow this protocol, which involves grinding the tissue in a mortar and pestle with liquid
nitrogen. Precipitate the proteins with 20% trichloroacetic acid (TCA) in prechilled acetone (–20°C). To remove
the plant phenols, rinse the pellet at least twice with cold acetone (–20°C) and air-dry samples in a vacuum
(Damerval 1986).
Reagents
■■
■■
■■
Protein precipitation solution
Wash solution
2-D sample solution
Protocol
ool protein precipitation and wash
C
solutions to –20°C and chill a mortar
with liquid nitrogen.
7
Centrifuge the solution at –20°C for
15 min at 35,000 × g and discard
the supernatant.
2
lace leaves in the mortar, add liquid
P
nitrogen, and grind the leaves
in the liquid nitrogen to a fine powder.
8
Add 2 ml of wash solution and suspend
the pellet.
9
Transfer the suspension into a shallow
ceramic shell and cover with perforated
Parafilm wrap.
4
5
6
80
Buffer Exchange (Desalting)
Bio-Rad’s Micro Bio-Spin columns are suitable for use with 1.5 or 2.0 ml microcentrifuge tubes and are
completely autoclavable. They accommodate volumes of 20–75 µl; volumes less than 20 µl may affect
recovery. The gel in the Micro Bio-Spin columns is suspended in either SSC buffer, pH 7.0, or Tris-HCl buffer,
pH 7.4. For 2-D electrophoresis, it is best to exchange the sample into the 2-D sample solution (7 M urea,
2 M thiourea, 4% CHAPS) using the following protocol. DTT and ampholytes are added after the buffer
exchange procedure.
Protocol
1
3
Methods
Transfer leaf powder into 20 ml protein
precipitation solution and incubate for
1 hr at –20°C. Stir solution occasionally.
Centrifuge the solution at –20°C for
15 min at 35,000 × g.
iscard the supernatant, add wash
D
solution, and suspend the pellet.
Incubate for 15 min at –20°C and stir
the solution occasionally.
Repeat steps 4 and 5 until the wash
solution turns from dark to light green.
10
1
ut the shell into a dessicator and
P
apply a vacuum until the pellet
(acetone powder) is dry.
ix 5 mg of sample powder with
M
~0.5 ml of 2-D sample solution
and incubate for 30 min at room
temperature. Vortex from time to time.
2
12
Centrifuge the solution at room
temperature for 15 min at >16,000 × g.
3
13
ollect the supernatant and perform
C
the protein assay.
11
Invert the column sharply several
times to resuspend the settled gel
and remove any bubbles. Snap off
the tip and place the column in a
2.0 ml microcentrifuge tube (included).
Remove the top cap. If the column does
not begin to flow, push the cap back on
the column and then remove it again to
start the flow. Allow the excess packing
buffer to drain by gravity to the top of
the gel bed (about 2 min). Discard the
drained buffer, then place the column
back into the 2.0 ml tube.
4
5
Place the column in a clean 1.5 or 2.0 ml
microcentrifuge tube. Carefully apply
the sample (20–75 μl) directly to the
center of the column. Application of
more or less than the recommended
sample volume may decrease
column performance.
Centrifuge the column for 2–4 min at
1,000 × g. Following centrifugation,
the purified sample is in the new
buffer. Molecules smaller than the
column’s exclusion limit are retained
by the column.
Centrifuge for 2 min in a microcentrifuge
at 1,000 × g to remove the remaining
packing buffer. Discard the buffer.
Apply the new buffer in 500 μl aliquots.
After each application, let the buffer
drain out by gravity, then centrifuge
the column at 1,000 × g for 1 min
to remove the buffer. Discard the
buffer from the collection tube.
Repeat as required. Three washes
result in >99% of the buffer exchanged.
Four washes result in >99.9% of the
buffer exchanged.
81
2-D Electrophoresis Guide
Chapter 8: Sample Preparation
Methods
Sample Quantitation (RC DC Protein Assay)
The RC DC protein assay is based on a modification of the Lowry protocol (Lowry et al. 1951) and is both
reducing agent compatible (RC) and detergent compatible (DC). Protein quantitation can be performed in
complex mixtures including 2-D sample solution. It involves addition of detection reagents to a protein solution
and subsequent measurement of absorbance at 750 nm with a spectrophotometer. Comparison to a standard
curve provides a relative measurement of protein concentration.
Microfuge Tube Assay Protocol (1.5 ml)
1
2
3
4
5
82
Add 5 μl of DC Reagent S to each
250 μl of DC Reagent A needed.
This solution is referred to as
Reagent A´. Each standard or sample
assayed requires 127 μl Reagent A´.
repare 3–5 dilutions of a protein
P
standard (0.2–1.5 mg/ml protein).
Use distilled or deionized water as
the diluent.
ipet 25 µl of protein standard
P
or sample into clean 1.5 ml
microcentrifuge tubes. Add 125 µl
of RC Reagent I into each tube and
vortex. Incubate the tubes for 1 min
at room temperature.
Add 125 µl of RC Reagent II into
each tube and vortex. Centrifuge
the tubes at 15,000 x g for 5 min.
Position the tubes with the cap
hinge facing outward.
6
7
Add 127 µl of Reagent A´ to each tube
and vortex. Incubate tubes at room
temperature for 5 min, or until the
precipitate is dissolved. Vortex.
Add 1 ml of DC Reagent B to each tube
and vortex immediately. Incubate at
room temperature for at least 15 min,
but no longer than 1 hr.
8
ead absorbance of each sample at
R
750 nm. The absorbances are stable
for at least 1 hr.
9
lot absorbance measurements as
P
a function of concentration for the
standards.
10
Interpolate the concentration of the
protein samples from the plot and
sample absorbance measurements.
Remove the tubes as soon as
centrifugation is complete. A small
pellet should be visible on the
hinge side of the tube. Decant the
supernatant. Reposition the tubes as
before. Briefly centrifuge again to bring
any remaining liquid to the bottom of
the tube. Use a micropipet to remove
the remaining liquid.
83
2-D Electrophoresis Guide
Methods
TABLE OF CONTENTS
CHAPTER 9
First-Dimension IEF
with IPG Strips
84
85
2-D Electrophoresis Guide
Chapter 9: First-Dimension IEF with IPG Strips
Tips for IEF
■■
■■
■■
■■
■■
IPG Strip Rehydration and Sample Loading
aster 2-D separation techniques using the
M
ReadyPrep™ 2-D starter kit (catalog #163-2105)
before using your own samples. The kit contains
premixed reagents, a standard sample, and a
detailed and optimized protocol, which allows
you to become familiar with the 2-D workflow and
techniques while validating the performance of
your 2-D system
hen preparing solutions, use clean and dust-free
W
vessels to avoid keratin contamination
Prior to their use in IEF, IPG strips must be
rehydrated (with or without sample) to their original
thickness with rehydration solution (Table 9.1),
which is often the 2-D sample solution
(see Chapter 8).
Tips for Rehydration and Sample Loading
■■
■■
se highly purified laboratory water
U
(conductivity <2 µS)
se deionized urea prepared with a mixed-bed ion
U
exchange resin to avoid protein carbamylation by
cyanate, which forms in old urea
■■
ehydrate IPG strips for 12 hr–overnight at 20°C
R
(or room temperature)
fter rehydration in a rehydration/equilibration tray,
A
rinse and blot the IPG gel strips to remove excess
rehydration solution before transferring to the
focusing tray; otherwise, urea may crystallize
on the surface of the IPG strips
Performing IEF
IPG Strip Rehydration in Rehydration/
Equilibration Trays Followed by IEF
The instructions in this chapter pertain to the use of
the PROTEAN® i12™ cell and accessories. For more
details about the components of this system and their
assembly and use, please refer to the PROTEAN i12
cell instruction manual (bulletin 10022069).
1
2
Table 9.1. Rehydration volumes and sample loads. Protein load recommendations are intended as a starting point, and the optimum amount
for the sample must be determined empirically. For narrow-range IPG strips, use more protein (proteins outside the range will not remain on the strip).
For single-pH-unit IPG strips, use up to 4–5 times more protein to improve the detection of low-abundance proteins.
Rehydration solution IPG Strip Length, cm
7
11 17 18 24
125 µl
200 µl
300 µl
315 µl
450 µl
Protein load
Coomassie (Brilliant) Blue
50–100 µg
100–200 µg
200–400 µg
200–400 µg
400–800 µg
Fluorescent stains
5–100 µg
20–200 µg
50–400 µg 50–400 µg
80–800 µg
Silver stains
5–20 µg
20–50 µg
50–80 µg
50–80 µg
80–150 µg
4 ml
5 ml
7 ml
7 ml
9 ml
Mineral oil
IEF with Gel-Side Up
The following protocol is for IPG strips that have
been rehydrated in the presence of sample
(in-gel sample loading).
Protocol
1
Protocol
oisten electrode wicks with deionized water.
M
They should be moist, not wet
o not heat urea-containing buffers to >37°C to
D
avoid protein carbamylation
Methods
3
Pipet the rehydration solution
(with or without sample, see Table 9.1
for volumes and protein loads) along
the center of the channel(s) of the
i12 rehydration/equilibration tray.
Take care not to introduce air
bubbles when expelling the solution.
sing forceps, remove the cover sheet
U
from the IPG strip, then gently place
the IPG strip gel-side down onto the
solution in the channel. Move the IPG
strip back and forth slightly to ensure
that the solution is distributed along its
length and that the strip is not sticking
to the bottom of the tray. Take care to
avoid trapping air bubbles beneath the
IPG strip.
Overlay each IPG strip with mineral oil
to prevent evaporation and precipitation
of urea during rehydration (see Table 9.1
for recommended volumes). Apply the
mineral oil to both ends of the channel
and allow it to flow toward the middle.
4
over the tray and leave it on a
C
level bench overnight (12–18 hr) for
complete rehydration.
5
ransfer the rehydrated IPG strips to
T
the focusing tray for IEF (see below).
2
3
sing forceps, remove the IPG strips
U
from the rehydration tray, remove
excess mineral oil, and place the
rehydrated IPG strips gel-side up
in the channels of the focusing tray.
Position the positive (+) ends of the
IPG strips against the positioning
stops in each channel.
Recommended: Wet the gel-side
up wicks (notched) with distilled or
deionized water and blot off excess
water. Use two wicks per IPG strip:
place a wick at each end of each
IPG strip.
osition the electrode assemblies
P
in the focusing tray and press
down on the green tabs to snap
the electrode assemblies into place.
Place the focusing tray with the
rehydrated IPG strips on the Peltier
platform and connect the electrodes
to the instrument.
4
Overlay each IPG strip with
mineral oil (see Table 9.1 for
recommended volumes).
5
Select or program the protocol(s)
and start the run.
IEF
Rehydration
With gel-side up
With sample
(in-gel sample loading)
Transfer IPG strips
to focusing tray
With gel-side down
Without sample
Transfer IPG strips
to focusing tray
With gel-side up
(cup loading)
Fig. 9.1. Sample loading.
86
87
2-D Electrophoresis Guide
Chapter 9: First-Dimension IEF with IPG Strips
Methods
Performing IEF (contd.)
IEF with Gel-Side Down
Cup Loading (IEF with Gel-Side Up)
IPG Strip Rehydration in the Focusing Tray Followed by IEF
The following protocol is for IPG strips that have
been rehydrated in the presence of sample
(in-gel sample loading).
This protocol is for IPG strips that have been
rehydrated in the absence of sample. Sample cups
offer an alternative method of sample loading, and
their use can often improve resolution, especially at
extreme pH ranges. The PROTEAN i12 sample cup
assembly consists of a sample cup holder that holds
1–12 disposable sample cups.
For rehydration and IEF in the focusing tray, place the IPG strip gel-side down on top of the rehydration solution
in the focusing tray. Rehydration can be programmed as a part of the IEF run, and the protocols can be
programmed next. Alternatively, the strips can be rehydrated independently and the protocol(s) started
when most convenient.
Protocol
1
2
3
4
5
6
7
osition the electrode assemblies in
P
the channels of the focusing tray and
press down on the green tabs to snap
the electrode assemblies into place.
1
sing forceps, place the rehydrated
U
IPG strips gel-side down in the
channels of the focusing tray.
Position the positive (+) ends of the
IPG strips against the positioning
stops in each channel.
2
3
Overlay each IPG strip with
mineral oil (see Table 9.1 for
recommended volumes).
lace the IPG strip retainers on top of
P
the IPG strips at both the positive and
the negative ends. Without IPG strip
retainers in place, gases formed
during electrolysis may lift IPG
strips off the electrodes, interrupting
electrical contact.
4
5
Select or program the protocol(s)
and start the run.
6
7
88
Position the electrode assemblies
in the focusing tray.
1
Protocol
Recommended: Wet the rectangular
(gel-side down) wicks with distilled or
deionized water and blot off excess
water. Use two wicks per IPG strip:
place a wick on top of each electrode.
lace the focusing tray on the Peltier
P
platform and connect the electrodes
to the instrument.
Protocol
sing forceps, place the rehydrated IPG
U
strips gel-side up in the channels of
the focusing tray. Position the positive (+)
end of the IPG strips against the
positioning stops in each channel.
Recommended: Wet the gel-side
up electrode wicks (notched) with
deionized water and blot off excess
water. Use two wicks per IPG strip:
place a wick at each end of each
IPG strip.
osition the electrode assemblies in
P
the focusing tray and press down on
the green tabs to snap the electrode
assemblies into place. Place the
focusing tray with the rehydrated IPG
strips on the Peltier platform, and
connect the electrodes to
the instrument.
repare the sample cup assembly by
P
placing the sample cups into the slots
of the sample cup holder corresponding
to the channel with the rehydrated
IPG strip.
lamp the sample cup assembly onto
C
the edges of the focusing tray, on top
of the IPG strips and next to either
electrode. Placement depends on the
pH gradient and the sample. In general,
focusing is most effective towards
the end of the IPG strip opposite the
site of the sample cup placement.
Use anodic sample cup placement
when using basic pH ranges or when
resolution of basic proteins is desired.
osition the IPG strip retainers on top
P
of the IPG strips at both the anode
and the cathode to maintain electrical
contact with the IPG strips. Without the
IPG strip retainers, gases formed during
electrolysis may lift the IPG strips
off the electrodes, interrupting
electrical contact.
6
Pipet the rehydration solution containing
the protein sample along the center of
the channel(s) of the focusing tray
(see Table 9.1 for recommended volumes
and protein loads). Do not introduce air
bubbles when expelling the solution.
2
Using forceps, remove the cover sheet
from the IPG strip, then gently place
the IPG strip gel-side down onto the
sample in the channel of the tray.
To ensure even rehydration, move the
IPG strip back and forth slightly to
distribute the solution along its length.
Check that no bubbles are trapped
beneath the strips and that some
rehydration solution extends beyond
the electrode contacts.
3
Rehydration in the focusing tray with in-gel
sample application can be programmed
as a part of the IEF
run or be performed separately.
7
To program rehydration as part
of the run:
a.Select or program the protocol(s)
for the lanes containing IPG strips
b.Program the global rehydration
conditions. If electrode wicks are
used, include a post-rehydration
pause to insert electrode wicks
when the rehydration step is
completed
lace the focusing tray with the IPG
P
strips on the Peltier platform and
connect the electrodes to
the instrument.
4
Immediately overlay each IPG strip with
mineral oil to prevent evaporation and
precipitation of urea during rehydration.
Apply the mineral oil to both ends
of the channel and allow it to flow
toward the middle. See Table 9.1 for
recommended volumes of mineral oil.
5
Start the run
For rehydration not programmed as part
of the run, leave the tray on the Peltier
platform or on a level bench overnight
(11–16 hr) for complete rehydration.
Start the run (perform IEF).
8
IEF
Rehydration
With sample
(passive or active)
c.
IPG strips remain
in focusing tray
With gel-side down
Fig. 9.2. Rehydration in the focusing tray.
oad 25–250 μl of sample into the
L
sample cups (larger volumes of dilute
samples of up to 400 μl may be loaded).
Overlay both the sample in the sample
cup and the IPG strip with mineral oil.
Select or program the protocol(s)
and start the run.
89
2-D Electrophoresis Guide
Chapter 9: First-Dimension IEF with IPG Strips
Methods
IEF Programming Recommendations
The protocols and settings described are for IEF using
the PROTEAN i12 IEF cell. Preprogrammed protocols
serve as convenient starting points for optimization of
IEF conditions (Tables 9.2 to 9.5).
■■
■■
■■
he recommended focusing temperature for most
T
samples is 20°C
or better sample entry, start IEF with a low voltage
F
gradient (200 V for 30–180 min) and limit current to
50 µA per IPG strip for the whole run
■■
ocusing time depends on gel length, pH gradient,
F
gel additives, and protein amount loaded.
Vertical streaking is often caused by overfocusing —
isoelectric precipitation (pI fallout) increases with
focusing time. For this reason, do not conduct firstdimension IEF for any longer than is necessary
fter completion of the IEF run, IPG strips should
A
be stored frozen at –80°C in rehydration trays
or immediately applied to a second-dimension
SDS-gel. Frozen IPG strips can be stored for
about 3–6 months
Table 9.2. Preprogrammed protocols for 7 cm ReadyStrip™ IPG strips.
Protocol Name
7 cm pH 3–10 R
7 cm pH 3–10 NL R
7 cm pH 4–7 R
7 cm pH 5–8 R
7 cm pH 3–10 G
7 cm pH 3–10 NL G
7 cm pH 4–7 G
7 cm pH 5–8 G
Voltage, V
Ramp
Time
Units
1
4,000 Rapid 15,000 Vh
2
1
7 cm pH 3.9–5.1
7 cm pH 4.7–5.9
1
1
90
HH:MM
4,000 Rapid Rapid 500
250 4,000 3
Rapid Gradual
500
4,000
Rapid Gradual
Rapid 15,000 Vh
10,000 Vh
0:15 HH:MMr
HH:MM
250 0:15 HH:MMr
1:00
HH:MM
2
4,000
4,000 3
1
250 4,000
4,000 1
1
Rapid Gradual
Rapid 500
4,000 Rapid 500
250 2
4,000
4,000 3
Rapid 500
2
3
Gradual
Rapid Gradual
Rapid 500
11 cm pH 3–10 G
11 cm pH 3–10 NL G
11 cm pH 4–7 G
11 cm pH 5–8 G
11 cm pH 3–6 R
20,000 11 cm pH 3–6 G
Vh
Hold
Rapid 11 cm pH 3.9–5.1
11 cm pH 4.7–5.9
Vh
0:15 HH:MMr
HH:MM
11 cm pH 5.5–6.7
11 cm pH 6.3–8.3
Vh
Hold
Time
Units
Rapid 26,000 Vh
2
1
Vh
11 cm pH 7–10 R
0:15 HH:MMr
HH:MM
11 cm pH 7–10 G
Vh
Hold
Hold
250 0:20 HH:MMr
1:00
HH:MM
8,000
8,000 3
Rapid Gradual
Rapid 4
1,500
8,000 1
1
HH:MMr
1:00
HH:MM
HH:MMr
1:00
HH:MM
HH:MMr
1:00
HH:MM
8,000 Rapid Rapid 40,000 Vh
Hold
29,000 Vh
750
Hold
250 0:20 HH:MMr
1:00
HH:MM
8,000
8,000 4
Rapid Gradual
750
2
3
Vh
0:20 8,000 1
32,000 250 Rapid Hold
8,000
1
Rapid Gradual
750
2
Vh
0:20 8,000 3
20,000 250 1
Rapid Hold
8,000
Rapid Gradual
750
2
3
Vh
0:20 8,000 1
20,000 250 Vh
Hold
Hold
8,000
3
Rapid 26,000 750
2
2
1:00
750
2
4
Hold
16,000 Ramp
8,000 4
1:00
16,000 Voltage, V
1
4
Hold
25,000 Step
2
1:00
20,000 11 cm pH 3–10 R
11 cm pH 3–10 NL R
11 cm pH 4–7 R
11 cm pH 5–8 R
Hold
500
1
Protocol Name
Hold
4
4
R = rapid, G = gradual
HH:MMr
1:00
2
2
7 cm pH 7–10 G
0:20 4,000 3
4
7 cm pH 7–10 R
250 4
7 cm pH 5.5–6.7
7 cm pH 6.3–8.3
Hold
4,000
2
7 cm pH 3–6 G
500
2
4
7 cm pH 3–6 R
Table 9.3. Preprogrammed protocols for 11 cm ReadyStrip IPG strips.
Step
Rapid Gradual
Rapid 750
29,000 Vh
Hold
R = rapid, G = gradual
91
2-D Electrophoresis Guide
Chapter 9: First-Dimension IEF with IPG Strips
Methods
IEF Programming Recommendations (contd.)
Table 9.5. Preprogrammed protocols for 24 cm ReadyStrip IPG strips.
Table 9.4. Preprogrammed protocols for 17 and 18 cm ReadyStrip IPG strips.
Step
Protocol Name
17 cm pH 3–10 R
17 cm pH 3–10 NL R
17 cm pH 4–7 R
17 cm pH 5–8 R
18 cm pH 3–10 R
18 cm pH 3–10 NL R
18 cm pH 4–7 R
18 cm pH5–8 R
17 cm pH 3–10 G
17 cm pH 3–10 NL G
17 cm pH 4–7 G
17 cm pH 5–8 G
18 cm pH 3–10 G
18 cm pH 3–10 NL G
18 cm pH 4–7 G
18 cm pH 5–8 G
17 cm pH 3–6 R
18 cm pH 3–6 R
1
2
1
18 cm pH 3–6 G
18 cm pH 3.9–5.1
18 cm pH 4.7–5.9
17 cm pH 7–10 R
18 cm pH 5.5–6.7
18 cm pH 6.3–8.3
18 cm pH 7–10 R
18 cm pH 7–10 G
92
Rapid 43,000 Vh
1,000
250 Rapid Gradual
Rapid 4
1,000
10,000 1
1
250 10,000
10,000 3
1
250 10,000
10,000 1
Rapid Rapid Gradual
Rapid 1,000
250 2
10,000
10,000 3
Rapid Gradual
1,000
2
3
Rapid 1,000
2
Rapid Gradual
Rapid 4
1,000
10,000 1
1
250 10,000
10,000 3
Rapid 1,000
2
4
R = rapid, G = gradual
10,000 10,000 3
2
17 cm pH 7–10 G
Units
4
17 cm pH 5.5–6.7
17 cm pH 6.3–8.3
Time
10,000
4
17 cm pH 3.9–5.1
17 cm pH 4.7–5.9
Ramp
2
2
17 cm pH 3–6 G
Voltage, V
Rapid Gradual
Rapid 1,000
Hold
0:30 HH:MMr
2:00
HH:MM
43,000 Vh
Protocol Name
Step
Voltage, V
Ramp
Time
Units
24 cm pH 3–10 R
24 cm pH 3–10 NL R
24 cm pH 4–7 R
24 cm pH 5–8 R
1
10,000 Rapid 60,000 Vh
24 cm pH 3–10 G
24 cm pH 3–10 NL G
24 cm pH 4–7 G
24 cm pH 5–8 G
Hold
32,000 Vh
24 cm pH 3–6 R
Hold
HH:MMr
2:00
HH:MM
24 cm pH 3–6 G
Vh
Hold
HH:MMr
2:00
HH:MM
24 cm pH 3.9–5.1
24 cm pH 4.7–5.9
Vh
Hold
HH:MMr
2:00
HH:MM
24 cm pH 5.5–6.7
24 cm pH 6.3–8.3
Vh
Hold
46,000 Vh
24 cm pH 7–10 R
Hold
0:30 HH:MMr
HH:MM
46,000 10,000 3
24 cm pH 7–10 G
Vh
Hold
Rapid Gradual
Rapid 4
1,500
10,000 1
1
250 10,000
10,000 3
1
250 10,000
10,000 1
Rapid Rapid Gradual
Rapid 1,500
250 2
10,000
10,000 3
Rapid Gradual
1,500
2
3
Rapid 1,500
2
Rapid Gradual
Rapid 4
1,500
10,000 1
2
2:00
250 10,000
4
0:30 63,000 1
1,500
2
4
0:30 50,000 2
0:30 32,000 2
1
250 2
10,000
10,000 3
4
Rapid 1,500
Rapid Gradual
Rapid 1,500
Hold
0:30 HH:MMr
2:00
HH:MM
60,000 Vh
Hold
44,000 Vh
Hold
0:30 HH:MMr
2:00
HH:MM
44,000 Vh
Hold
0:30 HH:MMr
2:00
HH:MM
70,000 Vh
Hold
0:30 HH:MMr
2:00
HH:MM
70,000 Vh
Hold
63,000 Vh
Hold
0:30 HH:MMr
2:00
HH:MM
63,000 Vh
Hold
R = rapid, G = gradual
93
2-D Electrophoresis Guide
Methods
TABLE OF CONTENTS
CHAPTER 10
Second-Dimension
SDS-PAGE
94
95
2-D Electrophoresis Guide
Chapter 10: Second-Dimension SDS-PAGE
IPG Strip Equilibration
Tips for SDS-PAGE
■■
■■
■■
■■
■■
nsure that gels have the same composition by
E
either using precast gels, which are manufactured
in lots and so are virtually identical, or hand
casting the gels at the same time in a multicasting chamber
ave time by preparing the overlay solution
S
and running buffers during the 10 min
equilibration incubations
ertical streaking on second-dimension gels is
V
often caused by gaps between the IPG strips and
the gels. Ensure that the second-dimension gel
has a straight and level top edge, and that the
IPG strip is in direct contact with the gel along its
entire length
hen preparing running buffers, make the solution
W
as specified in the protocol and do not titrate to a
pH. The ion balance is set by the concentration of
reagents; adjusting the pH alters this balance and
leads to undesirable results
Do not reuse running buffers
Methods
■■
■■
■■
■■
se 5–10 V per cm of gel for 10 to 30 min during
U
sample entry (until the sample has concentrated
at the starting point of the separation gel).
Then continue with the voltage setting
recommended in the instruction manual for
the electrophoresis system you are using
se the voltage setting recommended in the
U
instruction manual for the electrophoresis system
you are using; excessive voltage leads
to decreased resolution and distortions
hen running multiple cells, use the same
W
voltage for multiple cells as you would for one cell.
Be aware that the current drawn from the power
supply will double with two — compared to one —
cells. Use a power supply that can accommodate
this additive current and set the current limit high
enough to permit this additive function
o maximize reproducibility, maintain the
T
temperature of the electrophoresis buffer at
about 20°C with the help of a recirculating cooler
Equilibrate the IPG strips twice, each time for 10 min,
in two different equilibration buffers. Use disposable
rehydration/equilibration trays for this purpose.
Protocol
1
Reagents
Tris-HCl buffer (25 ml)
1.5 M Tris-HCl (pH 8.8)
Dissolve 4.55 g of Tris base in ~20 ml of deionized
or distilled H2O. Adjust the pH of the solution with
diluted HCl and adjust the volume to 25 ml with
distilled or deionized H2O.
2
3
Equilibration stock buffer (500 ml)
6 M urea, 30% (w/v) glycerol, 2% (w/v) SDS in 0.05 M
Tris-HCl buffer, (pH 8.8). Pre-prepared equilibration
buffers can also be purchased.
Combine 180 g of urea, 150 g of glycerol, 10 g of SDS,
and 16.7 ml of Tris-HCl buffer. Dissolve in deionized
distilled H2O and adjust the volume to 500 ml.
Store frozen.
4
lace one IPG strip gel-side up in each
P
channel of a rehydration/equilibration
tray, and fill the channels with
the recommended volume of
equilibration buffer.
Incubate with gentle agitation for
10 min, then decant.
ill the channels with the recommended
F
volume of equilibration buffer 2,
and incubate again for 10 min.
fter equilibration, remove the IPG
A
strips and briefly rinse with the
SDS-PAGE running buffer you will be
using. This step rids the IPG strip of
excess iodoacetamide and serves to
lubricate the IPG strip for placement
on the second dimension.
Equilibration buffer 1 (10 ml)
Add 100 mg of DTT to 10 ml of equilibration
stock buffer.
Equilibration buffer 2 (10 ml)
Add 400 mg of iodoacetamide to 10 ml of
equilibration stock buffer.
Table 10.1. Recommended equilibration volumes.
IPG Strip Length
7 cm 11 cm 17 cm 18 cm 24 cm
Equilibration buffer 1 2.5 ml 4 ml 6 ml 6 ml 8 ml
Equilibration buffer 2 2.5 ml 4 ml 6 ml 6 ml 8 ml
10 min is recommended for each equilibration step.
96
97
2-D Electrophoresis Guide
Chapter 10: Second-Dimension SDS-PAGE
Sealing IPG Strips onto SDS-PAGE Gels
In this stage, the equilibrated IPG strips are placed
on the top of polyacrylamide gels. This enables
smooth movement of the focused proteins into the
gel for separation by SDS-PAGE.
SDS-PAGE
Protocol
1
Reagents
Agarose solution (0.5% [w/v]): Suspend 0.5 g of
low-melting agarose (low electroendosmosis, EEO)
in 100 ml of SDS-PAGE running buffer, and dissolve
it in a boiling water bath or in a microwave oven.
Add a few crystals of bromophenol blue (or 100 µl of
1% bromophenol blue) to color the solution slightly.
The agarose solution can be aliquoted into sealed
1.5 ml or 2.0 ml plastic tubes, which can then be
melted individually in a 100°C heat block when needed.
2
3
Caution: Wear protective gloves, goggles, and a
lab coat when handling molten agarose. SDS in the
molten agarose can cause the solution to bubble
over. Molten agarose and the vessel containing it
can cause severe burns if not handled carefully.
Molecular weight standards: SDS-PAGE standards
can be applied to gels that have no reference lane.
Trim a thin filter paper to ~4 × 5 mm and pipet 10 μl of
SDS-PAGE standards onto the wick. Remove excess
solution with filter paper. Alternatively, use Precision
Plus Protein™ standard plugs, which can be used on
vertical 2-D gels with or without a reference well.
4
5
6
Buffers and Solutions
Position the second-dimension gel
cassette so that it is leaning slightly
backwards (approximately 30°
from vertical). Use AnyGel™ stands,
if available.
lace the equilibrated IPG strip (anodic
P
side on the left) onto the long plate with
the plastic backing against the plate.
Slide the strip between the plates using
a spatula to push against the plastic
backing. Ensure that the plastic backing
remains fully in contact with the long
plate and be careful not to damage the
gel with the spatula. Make sure the IPG
strip is positioned directly on top of
the second-dimension gel without any
bubbles in the interface between the
two gel surfaces.
Optional: Slip a wick soaked with
molecular weight standards or use a
Precision Plus Protein standard plug in
the slot in the gel sandwich next to or
overlapping an end of the IPG strip.
To secure the strip in place, overlay it
with molten agarose solution. Use warm
molten agarose, as hot agarose may
accelerate decomposition of the urea in
the equilibration buffer. Avoid trapping
air bubbles between the IPG strip and
second-dimension gel. Dislodge any
bubbles by tapping the plastic backing
on top of the strip.
tand the gel upright and allow
S
the agarose to set for 5–10 min
before loading the gel into the
electrophoresis cell.
This step requires the use of running buffer appropriate
for the gel chemistry you are using.
SDS-PAGE Running Buffer
(Tris-HCl and TGX™ formulations)
Prepare sufficient 1× Tris/glycine/SDS running buffer
to run the number of gels in the system selected:
1 L of 1× Tris/glycine/SDS (25 mM Tris, 192 mM
glycine, 0.1% SDS)
Tris base
Glycine
SDS
Distilled or deionized H2O 3.03 g
14.4 g
1.0 g
to 1 L
Alternatively, dilute 10× stock solution
(catalog #161-0732) to the desired volume.
Protocol
Perform SDS-PAGE according to the running
conditions specified for the electrophoresis system
you are using. In general:
1
2
3
4
98
Methods
Insert the gel cassettes in the
electrophoresis apparatus and fill the
buffer chamber(s) with SDS running
buffer. SDS running buffer temperature
should be kept constant at 20°C
if the chamber design allows for
external cooling.
Connect the electrophoresis cell
to a power supply and perform
electrophoresis at 5–10 V per cm of gel
until the sample has concentrated at
the starting point of the separation gel.
Then continue with the voltage settings
recommended by the instruction
manual for the electrophoresis
system you are using.
After electrophoresis, carefully open the
cassettes and use a spatula to separate
the agarose overlay, including the IPG
strip, from the polyacrylamide gel.
Carefully peel the gel from the
cassette and place it in a container
with fixative or staining solution,
depending on the staining procedure
used (see Chapter 11).
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2-D Electrophoresis Guide
Methods
TABLE OF CONTENTS
CHAPTER 11
Protein Detection
100
101
2-D Electrophoresis Guide
Chapter 11: Protein Detection
Total Protein Staining
Tips for Total Protein Staining
■■
■■
■■
■■
■■
■■
■■
tain gels at room temperature with gentle
S
agitation (for example, on an orbital shaker),
making sure the gel is completely covered
with stain solution at all times
se any convenient glass or plastic container
U
that is appropriate to the method chosen. Use
glass containers with silver staining methods
or with Flamingo™ stain. Use plastic trays with
SYPRO Ruby stain
se Bio-Rad’s Dodeca™ stainers for
U
high-throughput staining
■■
■■
■■
ear gloves during the staining process, and
W
handle gels only by the edges and corners.
Wet gloves with water or buffer before handling
the gel to keep the gel from sticking and tearing
se clean and dust-free containers for gel
U
staining. Place a lid on the container to avoid
contamination of the staining solution
se pure chemicals and highly purified water
U
(conductivity <2 μS)
hen performing gel staining with
W
fluorescent dyes, cover the staining tray
with foil during incubations
Methods
■■
Fluorescent dyes like Flamingo and Oriole™
fluorescent gel stains have a higher dynamic range
than Coomassie (Brilliant) Blue or silver staining
techniques and are, therefore, recommended for
quantitative protein analysis
els stained with fluorescent dyes can be
G
counterstained with Bio-Safe™ Coomassie stain
for further reference and to enhance sensitivity of
the Coomassie stain
ilver staining is not generally recommended
S
when protein spots will be identified by mass
spectrometry, though some formulations are
compatible with mass spectrometry at the
expense of promised sensitivity. Use Bio-Safe
Coomassie or fluorescent dyes like Flamingo
or Oriole instead
s an alternative to drying gels, seal them in
A
zip-top plastic bags in either water or, for long-term
storage, water with 0.005% sodium azide. Fill the
bag with water, insert the gel, expel the water,
and seal the bag
For more detailed instructions, refer to the
respective instruction manuals.
Bio-Safe Coomassie Stain
Instruction manual: bulletin 4307051.
2
3
Instruction manual: bulletin 10003321. Refer to
Table 11.1 for solution volumes.
Protocol
1
Protocol
1
Flamingo Fluorescent Gel Stain
Wash gels three times for 5 min each
in distilled or deionized H2O.
Remove water from staining container
and add Bio-Safe Coomassie stain to
completely cover the gel. Agitate for
at least 1 hr.
Rinse in distilled or deionized H2O for
at least 30 min. Stained gels can be
stored in water.
2
3
4
Place gel in a staining tray with fixing
solution (40% ethanol, 10% acetic acid).
Cover the tray and agitate gently for
at least 2 hr.
Pour off the fixing solution and add
1× stain solution (dilute 1 part Flamingo
fluorescent gel stain with 9 parts
deionized or distilled H2O). Cover the
tray and agitate gently. Stain for at
least 3 hr.
Optional background reduction:
Carefully pour off the stain solution
and replace with an equal volume of
0.1% (w/v) Tween 20. Cover the tray and
agitate gently for 10 min.
inse gel with deionized or distilled
R
H2O prior to imaging.
Table 11.1. Flamingo fluorescent gel stain.
Long-Term Storage of Stained Gels
Gels stained with a visible stain can serve as a
permanent record of the SDS-PAGE separation.
Stained gels may be stored indefinitely when dried
between cellophane sheets. To dry stained gels,
the gel is placed on a sheet of wet cellophane.
A second sheet of wet cellophane is carefully laid
over the gel with care taken not to introduce bubbles
or wrinkles. The gel, sandwiched between two
sheets of wet cellophane, is clamped into a frame
and allowed to dry.
The most common problem associated with drying
gels is cracking. Cracking is best prevented by
soaking the gel for at least 30 min in a 2% (w/v)
solution of glycerol in water prior to drying.
Alternatively, a commercially available gel-drying
solution may be used.
102
Fig. 11.1. 2-D gel stained with Bio-Safe Coomassie stain.
Gel size
Volume of fixing solution per gel
Volume of staining
solution per gel
Mini (8.6 × 6.8 cm)
100 ml
50 ml
Midi (13.3 × 8.7 cm)
200 ml
100 ml
Large (16 × 16 cm or 16 × 20 cm)
500 ml
250 ml
1,000 ml
500 ml
Larger (25.6 × 23 cm)
Fig. 11.2. 2-D gel stained with Flamingo stain.
103
2-D Electrophoresis Guide
Chapter 11: Protein Detection
Methods
Total Protein Staining (contd.)
Oriole Fluorescent Gel Stain
SYPRO Ruby Protein Gel Stain
Silver Stain Plus™ Kit
Instruction manual: bulletin 10017295. Refer to
Table 11.2 for solution volumes.
Instruction manual: bulletin 4006173. Refer to
Table 11.3 for solution volumes.
Instruction manual: bulletin LIT-442. Refer to
Table 11.4 for solution volumes and incubation times.
Protocol
Protocol
1
If using the 5 L configuration, prepare
the Oriole stain solution by adding
400 ml of methanol to the 1 L bottle
of diluent. Then add 10 ml of Oriole
fluorescent gel stain concentrate
and mix well by shaking.
Note: Do not fix or wash gel prior to staining. This will
make staining less sensitive.
2
3
Place gel in a staining tray with Oriole
fluorescent gel stain. Cover the tray and
agitate for ~1.5 hr. For best results, do
not leave gel in stain for more than 2 hr.
Rinse the gel in deionized distilled
H2O prior to imaging. Destaining is
not necessary.
1
Gel size
50 ml
100 ml
Large (16 × 16 cm or 16 × 20 cm)
250 ml
Larger (25.6 × 23 cm)
500 ml
ash the gel in one of the following
W
gel fixing solutions for 30 min:
■■
■■
■■
■■
2
3
Volume of staining solution per gel
Midi (13.3 × 8.7 cm)
■■
■■
Table 11.2. Oriole fluorescent gel stain.
Mini (8.6 × 6.8 cm)
Components:
4
10% methanol, 7% acetic acid
25% ethanol, 12.5% trichloroacetic acid
10% ethanol, 7% acetic acid
50% ethanol, 3% acetic acid
40% ethanol, 10% acetic acid
emove the wash solution and cover
R
the gel with SYPRO Ruby protein gel
stain. In general, use ~10 times the
volume of the gel. Using too little
stain will reduce sensitivity.
Stain the gel with continuous gentle
agitation for at least 3 hr for best
sensitivity. Specific staining can be
seen in 30–90 min. For convenience,
gels can be left in the stain solution
overnight (16–18 hr).
inse the gel in 10% methanol
R
(or ethanol), 7% acetic acid for
30–60 min to decrease background
fluorescence. Rinse the gel in water
before imaging.
■■
■■
■■
■■
■■
Fixative enhancer concentrate
Silver complex solution
Reduction moderator solution
Image development reagent
Development accelerator reagent
Empty 1L bottle for development accelerator reagent
2
3
Table 11.3. SYPRO Ruby fluorescent gel stain.
Gel size
Volume of staining solution per gel
8 × 108 cm
50 ml
16 × 20 cm
330 ml
20 × 20 cm 500 ml
4
Fig. 11.3. 2-D gel stained with Oriole stain.
Fig. 11.4. 2-D gel stained with SYPRO Ruby stain.
104
5
Step
Prepare the development accelerator
reagent solution. Add the entire
contents (50 g) of development
accelerator reagent to deionized
distilled H2O and bring volume up to 1 L.
Store at 4°C and use within 3 months.
Fixative step. Make fixative enhancer
solution by mixing 50% (v/v) reagentgrade methanol, 10% (v/v) reagent-grade
acetic acid, 10% (v/v) fixative enhancer
concentrate, and 30% (v/v) deionized
distilled H2O. After gel electrophoresis,
place gels in the fixative enhancer
solution with gentle agitation.
ater wash steps. Decant the fixative
W
enhancer solution from the staining
vessel. Rinse gels in deionized or
distilled H2O with gentle agitation.
Decant water and replace with fresh
rinse water and rinse. Decant rinse water.
Gel Thickness
0.75–1.0 mm
Time
Mini Gel
Large Gel
Fixative*
20 min
400 ml
800 ml
Water washes
10 min
400 ml
800 ml
Stain**
20 min
100 ml
300 ml
Stop
15 min
400 ml
400 ml
Protocol
1
Table 11.4. Silver Stain Plus.
Step
Gel Thickness
1.5–3.0 mm
Time
Mini Gel
Fixative*
30 min
400 ml
Large Gel
800 ml
Water washes
20 min
400 ml
800 ml
Stain**
20 min
100 ml
300 ml
Stop
15 min
400 ml
400 ml
*Gels may be left in this solution indefinitely prior to staining;
therefore, it is not necessary to carry out the entire procedure
directly following electrophoresis.
** S
tain until the desired intensity is reached. It may take at least
15 min before the first bands or spots become visible. Staining time
is dependent on the sample and quantity loaded.
Fig. 11.5. 2-D gel stained with Silver stain.
Staining step. To prepare staining
solution, add 35 ml of deionized or
distilled H2O to a beaker or flask with
a Teflon-coated stir bar. Add in the
following order: 5.0 ml of silver complex
solution, 5.0 ml of reduction moderator
solution, and 5.0 ml of image
development reagent. Immediately
before use, quickly add 50 ml of
development accelerator solution.
Stir well. Stain gels with gentle agitation.
Stop step. After the desired staining
is reached, place the gels in 5% acetic
acid solution to stop the staining
reaction. After stopping the reaction,
rinse the gels in high purity water for
5 min. Then the gels are ready to be
dried or photographed.
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Methods
TABLE OF CONTENTS
CHAPTER 12
In-Gel
Trypsin Digestion
106
107
2-D Electrophoresis Guide
Chapter 12: In-Gel Trypsin Digestion
Methods
Tryptic Digestion Protocol
This protocol for tryptic digestion of gel pieces (plugs)
excised from SDS-PAGE gels is derived from the
protocol described by Speicher et al. (2000). It can
be used in conjunction with any of the non-silver
stains described in this guide.
Reagents and Solutions
■■
Ammonium bicarbonate, NH4HCO3
■■
Acetonitrile
■■
Iodoacetamide
■■
■■
■■
■■
PLC-grade water (for example, Burdick and
H
Jackson AH365)
rifluoracetic acid (TFA) (for example,
T
Thermo Scientific 28904)
Octyl b-D-glucopyranoside (for example,
Sigma Aldrich 08001)
equencing-grade modified trypsin, porcine
S
(for example, Promega V5111)
Destaining buffer (50:50 ACN:0.2 M NH4HCO3)
Dissolve 158 mg of NH4HCO3 in 5 ml HPLC-grade
water and add 5 ml acetonitrile.
Destaining Gel Plugs from Silver-Stained
Gels (Pre-Treatment)
Gels that have been stained with a mass
spectrometry–compatible silver stain benefit
from an additional treatment to remove silver
metal by oxidation.
All materials used should be ACS reagent grade
or better.
Solution A (30 mM potassium ferricyanide)
To prepare 50 ml, dissolve 494 mg of potassium
ferricyanide [K3Fe(CN)6] in 50 ml of water. This solution
may be stored indefinitely at room temperature.
Solution B (100 mM sodium thiosulfate)
To prepare 50 ml, dissolve 791 mg of anhydrous sodium
thiosulfate [Na2S2O3] in 50 ml of water. This solution may
be stored for one year in a sealed bottle.
Reducing solution
Dissolve 555 mg of DTT in 3 ml of 50 mM NH4HCO3.
Alkylating solution
Dissolve 54 mg of iodoacetamide in 3 ml of 50 mM
NH4HCO3.
Extraction solvent
Combine 950 µl of 1% TFA with 50 µl of 1% octyl
D-glucopyranoside.
1
Mix Solutions A and B in a 1:1 ratio.
This is the silver destain solution.
2
lace each gel plug in a 0.5 ml or
P
1.5 ml plastic tube.
3
dd 50 µl of silver destain solution.
A
Incubate 20 min at room temperature.
4
sing a laboratory pipet, remove the
U
silver destain solution and add 50 µl
of fresh solution.
5
6
108
2
3
epeat steps 2 and 3 for a total
R
of three treatments. Following the
last incubation, remove the silver
destain solution.
Add 100 μl of destaining buffer to
the gel plug and incubate for 30 min.
Remove and discard the solution.
Digestion Protocol
1
Repeat step 1 two more times.
Add 400 μl of destaining buffer to
the gel plug and incubate overnight
at room temperature.
Reduction and Alkylation Protocol
2
3
This step is not necessary for 2-D gel plugs if they
have already been reduced and alkylated during the
sample preparation or equilibration steps.
1
Prepare the silver destain solution just prior to use.
It is good for only one use. Discard any excess.
Trypsin solution (20 μg/ml)
Dissolve 20 μg of trypsin in 1 ml of 50 mM NH4HCO3.
1
Protocol
50 mM NH4HCO3
Dissolve 79 mg of NH4HCO3 in 20 ml of water.
General Destaining Protocol
emove destaining buffer and
R
dehydrate the gel by adding 50 μl
of acetonitrile. Incubate 10 min
at room temperature and remove
excess solution (for example,
by aspiration).
2
Dry the gel piece for 30 min in a
laminar flow hood.
3
Add 100 μl of reducing solution to the
gel plug and incubate 30 min at room
temperature. Remove excess liquid.
4
dd 100 μl of alkylating solution to
A
the gel plug and incubate 30 min
at room temperature in the dark.
Remove excess liquid.
4
5
dd 50 μl of acetonitrile to the gel
A
plug and incubate for 10 min at room
temperature. Remove excess liquid
and proceed to digestion.
To the dried gel plug, add a volume
of trypsin solution equivalent to the
volume of the original hydrated plug
(1.5 mm plug = 3.4 μl).
Incubate at room temperature for
10 min (center of gel will change from
opaque to clear). If gel plugs aren’t
swollen, add a few more μl of trypsin
solution and incubate for an additional
10 min.
Add enough 50 mM NH4HCO3
to cover the gel plug (~10 μl).
Incubate at 37°C for at least 3 hr.
Extraction Protocol
1
2
3
4
Remove trypsin solution from the
gel plug, and store it in another vial.
To the gel plug, add 2–8 µl of extraction
solvent. For MALDI-MS analysis,
keep this volume as small as possible
(2–3 µl). For LC-MS analysis, add 8 µl.
Incubate 30 min at room temperature.
Combine extraction solvent with
trypsin solution.
Proceed with the procedure
described below.
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2-D Electrophoresis Guide
Troubleshooting
TABLE OF CONTENTS
PART III
Troubleshooting
110
111
2-D Electrophoresis Guide
Troubleshooting
Isoelectric Focusing
ProblemCause
Isoelectric Focusing (contd.)
Solution
Initial low or zero current
Poor contact between IPG strips Make sure that the gel side of the IPG strip
and electrodes
is in contact with the electrode
For the gel-side down configuration with
the PROTEAN® i12™ cell, use the IPG
strip retainers
Incomplete wetting of electrode wicks
Wet the electrode wicks with distilled or
deionized H2O until they are damp, but not soaking wet
Incomplete IPG strip rehydration
Check the rehydration volumes and times for the lengths of IPG strips used
No current in any lane
No contact between the Make sure that:
■ The electrode assembly is properly
electrode assembly and IPG strips seated in the focusing tray
■ The IPG strips are positioned correctly, (for example, that the gel is in direct contact with the electrode)
No contact between the Make sure that:
■ The gold contact pin of the negative (–) electrode assembly and instrument assembly is in direct contact with the cathode bar on the instrument
■ The positive (+) assembly is completely inserted into the anode of the instrument
Voltage does not
High levels of ionic contaminants Keep salt concentrations under 40 mM; increase beyond initial
in sample solution (optimum salt
if necessary, desalt the sample
low voltage steps
concentration is ~10 mM, though (for example with Micro Bio-Spin™ 6
up to 40 mM can be tolerated)
columns or the ReadyPrep™ 2-D cleanup kit)
Salt collects in electrode wicks, so
replace electrode wicks from time to time
(every 2 hr) during the initial low-voltage
steps. Several hours may be needed for
ionic contaminants to leave IPG strips
Voltage does not reach
Programmed voltage is too
programmed value, high for the pH range and
or maximum voltage is
length of IPG strip
reached very slowly.
Lower the voltage maximum set for the focusing step; the conductivity and the
length and type of IPG strip determine the
voltage maximum that can be reached
Note: good focusing
may be obtained even
if programmed voltage
is never reached
Ampholyte concentration is too high. Up to 1% (v/v)
Bio-Lyte® ampholytes may
be used, but ampholytes
increase conductivity;
therefore, voltage will be lower
with increasing concentrations
112
Lower the ampholyte concentration
ProblemCause
Solution
Excess sample during rehydration Use correct rehydration volumes for did not enter gel, or IPG gels are
the lengths of the IPG strips used
overswelled with excess sample
Voltage is too high for the IPG strip size and pH gradient
Program Vh for the IEF step to ensure
complete focusing of the sample
Large fluctuations in IPG strips contain poorly
voltage and current rehydrated regions, or IPG strips
have dried out during the run
Check rehydration volumes and times
Burning of strips Current limit is too high
Use a current limit of 50 µA/IPG strip
IPG strips have dried out Make sure that the IPG strips are covered
with mineral oil or equivalent
Make sure that the rehydration solution is evenly distributed during rehydration and that the IPG strips are completely covered with mineral oil
Electrode wicks are too wet or
Wet the electrode wicks with distilled contain incorrect electrode solution or deionized H2O until they are damp, not soaking wet
Incorrect rehydration
solution composition
Check the composition of the
rehydration solution
Sample is leaking from
Cup positioning is incorrect
the sample cups
When positioning the cup holder, make sure that it clicks into place at the edges of the focusing tray
The cup is positioned in an
area of the IPG strip that is
not completely rehydrated
Make sure that the IPG strips are
rehydrated evenly and thoroughly
The cup is malfunctioning
Replace cup
SDS-PAGE
ProblemCause
Solution
Low or zero current, and samples do not migrate into the gel Remove the tape
With a precast gel, the tape
at the bottom of the gel cassette
was not removed
Insufficient buffer in the inner or outer buffer chamber
Fill the inner and outer buffer chambers to ensure that the IPG well is
completely covered
Electrical disconnection Check the electrodes and connections
Running time slower or
faster than expected
Incorrect running buffer
concentration or type
Check the buffer composition and type
Leaking from inner Incomplete gasket seal buffer chamber
Wet the gasket with running buffer
before use
■
Improper assembly of the gel
into the electrode/ companion assembly
■
Ensure that the top edge of the short plate fits under the notch at the top
of the gasket
Ensure that the top of the short plate touches the green gasket
113
2-D Electrophoresis Guide
Troubleshooting
Total Protein Staining
Total Protein Staining (contd.)
ProblemCause
Solution
ProblemCause
Solution
Spots not visible (see 2-D No protein in the gel
Gel Evaluation, below)
Use another staining method to confirm that there is protein in the gel
Check the instrument manual for troubleshooting information, or contact the imaging instrument manufacturer
Speckles or blotches in Particulate material from reagents, the gel image
staining tray, dust, or gloves
Clean the staining trays and other
equipment thoroughly with laboratory glassware cleaner
Malfunctioning imaging system or incorrect
imaging parameters
Poor staining Insufficient protein in the gel
load sensitivity
Repeat the experiment with a
higher protein quantity
Dirty staining trays (for example, with silver staining)
Clean the staining trays and other equipment thoroughly with laboratory glassware cleaner
Insufficient stain volume Follow the recommendations for stain volume appropriate to the gel size
Increase staining time
Insufficient staining time
Use dust-free gloves, and handle gels
only by the edges
Uneven staining
Insufficient shaking during staining Agitate the gel during staining
Gel shrinkage
Some gel shrinkage occurs during staining Transfer the gel to water
2-D Gel Evaluation*
Reuse of staining solution
To ensure quantitative reproducibility
of a 2-D experiment, never reuse staining solution
ProblemCause
High or uneven Dirty equipment or staining trays
background staining
Clean the staining trays and other equipment
thoroughly with laboratory glassware cleaner
Across the gel Insufficient sample was loaded
Check the sample concentration by
protein assay
Check that the protein assay is functioning
properly and that it is not responding to interfering substances in your sample
Insufficient sample entered the IPG strip
Start IEF at a low field strength
Check that the orientation of electrical connections
Too much time in staining solution Restrict the time in staining solution as recommended
114
Limit exposure of gels and staining
solution to open air
Solution
No Spots or Fewer Spots
than Expected
Wash the gel in water or respective destaining solution for >30 min
Reagent impurities
Make sure that the water and reagents used for staining are of the highest
possible quality
Diffuse, uneven Insufficient washing background in silver-stained gel
Perform more washing steps. Use purified
laboratory water and clean staining trays
Do not place too many gels in one tray.
Fully immerse the gels in the staining solution; they should not stick to the
staining tray
Increase the solubility strength of the
2-D sample solution; insoluble proteins
will not enter the IPG strip
Insufficient fixative (some uneven Apply a longer fixing procedure
background stain is normal when
using a silver stain. Due to migration
of different chemicals and ions
into the gels, some regions can
be stained with different colors
or intensities)
Contaminant(s) in the agarose overlay solution Make sure that the IPG strips are in the correct orientation in the focusing tray
Failure of detection reagentsRun a lane of unstained standards adjacent
to the second-dimension separation. If the
standards are not detected, check the
expiration dates and the formulations of
all detection reagents
Staining method not sensitive enough
See Chapter 3 for sample loading
recommendations dependent on the staining technique used
Poor protein transfer from IPG strip to SDS-gel
Perform the first stage of SDS-PAGE at
low voltage (50 V) for >20 min until the
bromophenol blue front enters the
separation gel (time depends on gel size)
* Also refer to Berkelman et al. (2004) and Bio-Rad Laboratories (2005).
Prepare fresh overlay solution
115
2-D Electrophoresis Guide
Troubleshooting
Horizontal Streaking (contd.)
2-D Gel Evaluation (contd.)
ProblemCause
Solution
No Spots or Fewer Spots
than Expected
In high molecular Sample may have undergone
weight regions
proteolysis prior to IEF
Include appropriate protease inhibitors
and keep the sample on ice or in a cold
room during sample preparation
Insufficient equilibration Incubate IPG strips in sufficient volumes
of each equilibration buffer for up to
15 min with mild agitation
Poor entry of high molecular Use active sample loading in the focusing
weight proteins during rehydration tray or cup loading
(the pore size of the acrylamide in
the IPG strip is very small during
the early stages of rehydration) Poor entry of high molecular weight proteins into the
second-dimension gel
Increase equilibration time (2 × 15 min)
ProblemCause
Solution
Across the entire gel Treat the sample with a nuclease
DNA contamination Make sure that the nuclease is active and
that digestion is adequate; a very viscous
sample implies that nuclease treatment
has failed
Incomplete focusing
Optimize the sample focusing time by or overfocusingrunning a time course. For example,
run the sample on 6 IPG strips and
remove an IPG strip at each time point
(20 kV-hr, 30 kV-hr, 40 kV-hr, etc.)
Incomplete IPG strip rehydration Check the rehydration volumes and times
for the lengths of IPG strips used
Partial Incomplete IPG strip rehydrationCheck the rehydration volumes and
times for the lengths of IPG strips used
If the sample appears unevenly distributed,
or if areas of the IPG strip are not wetted
with sample, slide the IPG strip back and
forth several times along the length of the
channel in the focusing tray
Horizontal Streaking
Across the entire gel Protein overloading Use less sample
Perform prefractionation to enrich the
protein of interest and lower the amounts
of other abundant proteins
Use a longer IPG strip and larger gel size
to allow for a greater protein load
Proteins are not properly and Solubilize proteins completely using a
stably solubilized strong chaotropic extraction reagent.
The concentrations of urea, thiourea,
detergents, carrier ampholytes, and DTT
are also critical. Every sample type typically
requires a new sample preparation method
Regional Protein overloading Use less sample
Perform prefractionation to enrich the
protein of interest and lower the relative
amounts of other abundant proteins
Use a longer IPG strip and larger gel size
to allow for a greater protein load
In the basic range of the gel
Depletion of DTT in the basic range of the IPG strip
Treat the sample with the ReadyPrep
reduction-alkylation kit prior to IEF
Allow sufficient time for full denaturation
and solubilization; for example, incubate
the sample in the solubilization solution at
room temperature for 1 hr before applying
it to the IPG strip
116
117
2-D Electrophoresis Guide
Troubleshooting
Horizontal Streaking (contd.)
Vertical Streaking (contd.)
ProblemCause
Spots
Solution
Incomplete IEF Optimize the sample focusing time by
running a time course. For example,
run the sample on 6 IPG strips and
remove an IPG strip at each time point
(20 kV-hr, 30 kV-hr, 40 kV-hr, etc.)
ProblemCause
At one end of the gel (cup loading)
Intermittent Contaminants such as salts, ionic detergents (for example, SDS), peptides, nucleic acids, lipids, polysaccharides,
phenolic compounds
Use appropriate contaminant removal
techniques, such as treatment with
the ReadyPrep 2-D cleanup kit
Solution
Protein aggregation or
Dilute the sample to 3–5 µg/µl for
precipitation caused by cup loading
too much protein or sample
loading problemsPerform a protein assay prior to IEF to
ensure correct protein load. The total
amount of protein that should be loaded
onto an IPG strip depends on the length
of the strip and the stain that will be used
to visualize the results
Load the sample using in-gel sample loading
Prolong the time on the initial low-voltage
steps and increase the voltage gradually
Field strength used for sample loading is too high
Reduce the field strength to ~10 V/cm
IPG strip length
Poor protein solubilityIncrease the solubilizing strength of
2-D sample solution
Isolated streaking Improper rehydration of IPG stripCheck the rehydration volumes and times
for the lengths of IPG strips used
If the sample appears unevenly distributed,
or if areas of the IPG strip are not wetted
with sample, slide the IPG strip back and
forth several times along the length of the
channel in the focusing tray
Vertical Streaking
Across the entire gel 118
Leaking of the upper buffer Prior to inserting the gel(s) into the vertical
reservoir (cathode) of the vertical electrophoresis cell, wet the gaskets of the
electrophoresis unitelectrophoresis chamber with water or
use a small amount of vacuum grease
Incomplete equilibration
Increase equilibration time to 15 min
Old DTT and iodoacetamide preparations used in equilibration
Use fresh reagents for the equilibration step
Point streaking (handcast gels)
Dust or other particles in the
gel solutions
Filter gel solutions through a 0.45 μm
membrane and into a dust-free container
Vertical streaks connected to a spot
Insufficient binding of SDS
to protein
Check the SDS concentration (>1%) in the
equilibration solution
Increase equilibration time:
equilibrate IPG strips for 2 × 15 min
Incorrect pH in resolving gel buffer; Ensure that the pH of the Tris buffer used
incorrect pH decreases mobility for gel casting is 8.8
of protein-SDS complexes and
causes vertical streaks Buffer leakageEnsure that the upper buffer reservoir
is not leaking
119
2-D Electrophoresis Guide
Troubleshooting
Vertical Streaking (contd.)
Vertical Streaking (contd.)
ProblemCause
Solution
ProblemCause
Twin vertical spots or vertical doublets Make sure that the focused IPG strip is
in full contact with the gel
Blank stripes near pH 7
Improper placement of the IPG
strip onto the gel Temperature gradient in the gelLower the power settings for the seconddimension SDS-PAGE run, especially when
using cells that provide only one-sided
cooling of the gel
Use a better circulation system to improve
heat dissipation during a run
Blank vertical stripes Air bubble trapped in the agarose Ensure that the 2-D gel has a straight,
that joins the IPG strip to the top level top edge and that the IPG strip is
of the gelin direct contact with the 2-D gel along its
entire length. Squeeze out air bubbles
by pressing on the plastic backing of
the IPG strip
Use a 0.5% agarose overlay solution to
prevent the IPG strip from coming loose
or moving. To minimize the number of
bubbles in the overlay, melt the agarose
overlay solution completely prior to loading
120
Insufficient rehydration of a region of the IPG strip, or tears resulting from improper handling, resulting
in the absence of focused protein in that region
Make sure that the IPG strip is not sticking
to the bottom of the rehydration tray
Focusing of an amphoteric nonprotein contaminant
(for example, phospholipid or
HEPES) prevents protein focusing
around the pI of the contaminant
Apply sample cleanup
Excessive DTT (>50 mM) in the IPG sample solution
Solution
Lower the amount of DTT in the
rehydration solution
Blank stripes at the Salt buildup
electrodes, especially at the cathode
Remove ionic contaminants from the
samples with Bio-Rad´s ReadyPrep 2-D
cleanup kit or by desalting
Blank vertical regions Interfering substances; impurities in the rehydration/sample solution
Remove contaminants from the samples
with the ReadyPrep 2-D cleanup kit or
by desalting
Use high-quality reagents and chemicals
for electrophoresis to minimize the risk
of impurities. Replace chemicals of
questionable or unknown shelf life, origin,
or quality, as these products can also
contribute to poor 2-D results
Air bubble trapped in the agarose Ensure that the 2-D gel has a straight,
that joins the IPG strip to the top level top edge and that the IPG strip is
of the gelin direct contact with the 2-D gel along
its entire length. Squeeze out air bubbles
by pressing on the plastic backing of
the IPG strip
Use a 0.5% agarose overlay solution to
prevent the IPG strip from coming loose or
moving. To minimize the number of bubbles
in the overlay, melt the agarose overlay
solution completely prior to loading
Check the integrity of rehydrated IPG
strips prior IEF
121
2-D Electrophoresis Guide
Troubleshooting
ProblemCause
Solution
Other Problems
Wavy spots Insufficient overlay solution used Overlay the gel with water-saturated
in gel castingbutanol (n-butanol, l-butanol, or t-butanol)
or t-amyl alcohol immediately after gel
casting. These ensure that the gel has a
clean, straight top edge
Use precast gels
Use the overlay recommended by the
manufacturer of the electrophoresis cell
Localized wavy Problems with casting second-
disturbance of spots
dimension acrylamide gel: not evenly polymerized,
gel cassette leaking, etc.
Optimize the APS and TEMED
concentrations
Degas solutions prior to the addition
of APS/TEMED
Perform casting at room temperature,
warming the glass plates if necessary.
Be aware that the polymerization process
is temperature dependent. If the
temperature is too low, polymerization
may be compromised
Use precast gels
Known proteins appearing Protein carbamylation
Do not prepare samples too far ahead
as multiple spots or at the of time in urea
wrong position
Do not expose urea-containing samples to
high pH or temperatures that exceed 30°C
Protein oxidation Increase DTT concentration
Protein proteolysis Add protease inhibitors, perform
(during sample preparation)manipulations as quickly as possible,
and keep solutions as cold as possible
For further help or advice, please contact the Bio-Rad Technical Support department. In the United States, the Technical Support department
is open Monday–Friday, 5:00 AM–5:00 PM, Pacific time.
Phone: 1-800-424-6723
Fax: 1-510-741-5802
Email: [email protected] (for U.S. and international customers)
Online technical support and worldwide contact information are available at www.consult.bio-rad.com.
122
123
2-D Electrophoresis Guide
Appendices
TABLE OF CONTENTS
PART IV
Appendices
124
125
2-D Electrophoresis Guide
Appendices
Appendix A
Glossary
%CCross-linker concentration; weight percentage of cross-linker in a polyacrylamide
gel. Effective pore size of a gel is a biphasic function of %C
%TMonomer concentration (acrylamide + cross-linker) in a gel (in g/100 ml).
Effective pore size of a gel is an inverse function of %T, and gels can be made
with a single, continuous %T throughout the gel (single-percentage gels), or they
can be cast with a gradient of %T through the gel (gradient gels)
2-D electrophoresisTwo-dimensional electrophoresis. Proteins are separated first according to
isoelectric point (pI) by isoelectric focusing (IEF) and then according to size by
SDS-PAGE, yielding a two-dimensional protein map of spots
2-MercaptoethanolReducing agent used for cleavage of intra- and intermolecular disulfide bonds to
achieve complete protein unfolding and to maintain all proteins in a fully reduced
state. Also known as b-mercaptoethanol or BME
AcrylamideMonomer used with a cross-linker to form the matrix used for separating proteins
or small DNA molecules
Ammonium persulfate
(APS)
Initiator used with TEMED (catalyst) to initiate the polymerization of acrylamide and bisacrylamide in making a polyacrylamide gel; (NH4)2S2O8
Comb Object used to cast wells in an agarose or acrylamide gel. In PAGE applications,
square-bottom combs are inserted into the gel sandwich before polymerization
to form square-bottomed wells
Coomassie (Brilliant) Anionic dye used in the total protein staining of gels and blots and that comes in Bluetwo forms: Coomassie (Brilliant) Blue G-250 differs from Coomassie (Brilliant) Blue
R-250 by the addition of two methyl groups
Criterion™ cells, Family of Bio-Rad products used for midi-format vertical electrophoresis;
blotters, and gelsincludes the Criterion and Criterion™ Dodeca™ cells, Criterion blotter, and
Criterion precast gels
Cross-linker Molecule (for example, bis-acrylamide) used to link polymerizing monomer
molecules together to form a netlike structure within the gel. The holes in the nets
are called the pores, and the pore size is determined in part by the cross-linker
concentration. The pores may or may not sieve the macromolecules
Cup loadingApplication of protein sample onto IPG strips through sample cups applied to the
strips; can improve resolution at extremes of a pH gradient and improve uptake of
basic proteins
AmpholyteAmphoteric molecule that exists mostly as a zwitterion in a certain pH range.
Ampholytes are used to establish a stable pH gradient for use in isoelectric focusing
DC™ assay kit
Amphoteric
DepletionReduction in the amount of high-abundance proteins relative to
low-abundance proteins
Containing both acidic and basic groups
AnodePositively charged electrode. Negatively charged molecules (anions) move towards
the anode, which is usually indicated by the color red
Bio-Rad’s detergent-compatible protein assay kit
Anionic dye Negatively charged compound used as a stain; used in blotting to stain proteins
immobilized on membranes such as nitrocellulose or PVDF
Discontinuous Electrophoresis gel system that uses different buffers and sometimes
buffer systemdifferent buffer compositions to focus and separate components of a sample.
Discontinuous systems typically focus the proteins into tighter bands than
continuous gel systems, allowing larger protein loads
Antibody Immunoglobulin (Ig); protein produced in response to an antigen, which specifically
binds the portion of the antigen that initiated its production
Disulfide bondChemical bond joining two sulfur atoms; commonly found in proteins,
contributing to their secondary and tertiary structures
Assay Dithiotheithol (DTT)Reducing agent used for cleavage of intra- and intermolecular disulfide
bonds to achieve complete protein unfolding and to maintain all proteins in a fully
reduced state
Analysis of the quantity or characteristics of a substance
Background Nonspecific signal or noise that can interfere with the interpretation of valid signals
Bio-Spin ® columnsFamily of Bio-Rad sample preparation products that includes the Bio-Spin® 6 and
Micro Bio-Spin™ 6 columns; used for buffer exchange and desalting applications
Bis or bis-acrylamide A common cross-linker used with acrylamide to form a support matrix;
N,N'-methylene-bis-acrylamide
Blot Immobilization of proteins or other molecules onto a membrane, or a membrane
that has the molecules adsorbed onto its surface
Bromophenol blue
Common tracking dye used to monitor the progress of electrophoresis
Carrier ampholytesHeterogeneous mixture of small (300–1,000 Da) polyamino-polycarboxylate
buffering compounds that have closely spaced pI values and high conductivity.
Within an electric field, they align according to pI to establish the pH gradient
CathodeNegatively charged electrode. Positively charged molecules (cations) move toward
the cathode, which is usually indicated by the color black
Chaotropic agentChemical that disrupts inter- and intramolecular interactions (for example, urea
and thiourea)
126
CHAPSZwitterionic detergent (having both positively and negatively charged groups
with a net charge of zero) that is widely used for protein solubilization for IEF and
2-D electrophoresis; 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate
Electrophoresis
Movement of charged molecules in a uniform electric field
EquilibrationPreparation of protein separated in an IPG strip for second-dimension SDS-PAGE;
reduces and alkylates sulfhydryl groups and saturates proteins with SDS
EXQuest™ spot cutter
Bio-Rad’s brand of spot cutter
FractionationSeparation of a sample into discrete parts for separate analysis; may improve
detection of low-abundance proteins and reduce sample complexity
GlycineAmino acid used as the trailing or slow ion in SDS-PAGE according to Laemmli
(Laemmli, 1970)
Gradient gel Gel with gradually changing monomer concentration (%T) in the direction of
migration. In SDS-PAGE, gradients are used to separate wider molecular weight
ranges of molecules than can be separated with single-percentage gels
Immobilized pH Strips in which buffering groups are covalently bound to an acrylamide gel
gradient (IPG) stripsmatrix, resulting in stable pH gradients. This eliminates problems of gradient
instability and poor sample loading capacity associated with carrier
ampholyte–generated pH gradients
127
2-D Electrophoresis Guide
Appendices
Immunoblotting Blot detection by antibody binding
Immunodetection Detection of a molecule by its binding to an antibody
In-gel sample application Sample application to the IPG strip during IPG strip rehydration; may be passive
(In-gel rehydration) or active (in the presence of a low applied voltage)
Ionic strength Measure of the ionic concentration of a solution that affects its resistance
Isoelectric
focusing (IEF)
Electrophoresis technique that separates proteins according to their
isoelectric point (pI)
Isoelectric point (pI)pH value at which a molecule carries no net electrical charge, or at which the
negative and positive charges are equal
Leading ionIon in a discontinous buffer system with a greater mobility, typically Cl (chloride ion)
–
MicroRotofor™ cells and kits
Family of Bio-Rad sample preparation products, including the MicroRotofor
liquid-phase IEF cell and MicroRotofor cell lysis kits
Monomer Unit that makes up a polymer (acrylamide is a monomer that is polymerized
into polyacrylamide)
Mini-PROTEAN ® cells Family of Bio-Rad products used for mini-format vertical electrophoresis;
and gelsincludes the Mini-PROTEAN Tetra and Mini-PROTEAN® 3 Dodeca™ cells
and Mini-PROTEAN precast gels
Molecular weight markers
Mixtures of well-characterized or recombinant proteins used to help monitor
separation as well as estimate the size of the proteins separated in a gel
Ohm’s LawDescribes the mutual dependence of three electrical parameters
(V, voltage; I, current; R, resistance): V = I × R
PAGE Polyacrylamide gel electrophoresis, a common method of separating proteins
based on molecular weight
PDQuest™ software
Bio-Rad’s 2-D gel analysis software
PolyacrylamideAnticonvective, sieving matrix used in gel electrophoresis. Polyacylamide gels
are cast using mixtures of acrylamide monomers with a cross-linking reagent,
usually N,N'-methylenebisacrylamide (bis), both dissolved in buffer
Polyacrylamide gel Electrophoresis technique that uses polyacrylamide as the separation medium
electrophoresis (PAGE)
PowerPac power supplies
™
Family of Bio-Rad power supplies
Power supply Instrument that provides the electric power to drive electrophoresis
and electrophoretic blotting experiments
Precision Plus Protein™ Bio-Rad’s family of recombinant molecular weight markers
standards
PROTEAN ® cellsFamily of Bio-Rad products used for large-format vertical electrophoresis
and isoelectric focusing; includes PROTEAN II xi, PROTEAN II XL,
PROTEAN® Plus Dodeca™ cells, and the PROTEAN® i12™ IEF cell
Protein enrichment technology that operates on the principle of dynamic range
ProteoMiner™ beads, reagents, and kitsreduction; uses a bead-based library of combinatorial peptide ligands to enrich the
amounts of medium- and low-abundance proteins relative to high-abundance proteins
Rf value Relative distance a protein has traveled compared to the distance traveled by
the ion front. The Rf value is used to compare proteins in different lanes and
even in different gels. It can be used with standards to generate standard curves,
from which the molecular weight or pI of an unknown may be estimated
Running bufferBuffer that provides the ions for the electrical current in an electrophoresis run.
It may also contain denaturing agents. The running buffer provides the trailing ions
in discontinuous electrophoresis
Sample solution Solution in which a sample is prepared or suspended prior to loading onto
an IPG strip
Sodium dodecyl sulfate Separation of molecules by molecular weight in a polyacrylamide gel matrix in
polyacrylamide gel the presence of a denaturing detergent, such as sodium dodecyl sulfate (SDS).
electrophoresis SDS denatures polypeptides and binds to proteins at a constant charge-to-mass-ratio.
(SDS-PAGE)In a sieving polyacrylamide gel, the rate at which the resulting SDS-coated proteins
migrate in the gel is relative only to their size and not their charge or shape
Sodium dodecyl sulfate (SDS)
Anionic detergent that denatures proteins and binds to polypeptides in a constant
weight ratio of 1.4 g/g of polypeptide (SDS:polypeptide)
Stain-free technologyProtein detection technology involving UV-induced additive that modifies protein
tryptophan residues. Continued exposure to UV light causes fluorescence of the
modified proteins, which are then detected by a CCD imager. Sensitivity of this
technique is generally equal to or better than Coomassie staining
Stained standards Mixture of molecular weight marker proteins that have covalently attached dye
molecules; the bands are visible during electrophoresis and transfer
Standard Collection of molecules with known properties, such as molecular weight,
isoelectric point, or concentration. Often used to create standard curves,
from which the properties of an unknown may be determined
TGX™
Bio-Rad’s Tris-glycine extended shelf life precast gels
Total protein stain Reagent that binds nonspecifically to proteins; used to detect the entire protein
pattern on a blot or gel
Trailing ionIon in a discontinous buffer system with a lower mobility, typically glycinate
Tris Organic component of buffer solutions that has an effective buffering range of
pH 7.0–9.2; tris(hydroxymethyl)aminomethane
Triton X-100Nonionic detergent widely used for protein solubilization
(for IEF and 2-D electrophoresis)
Tween 20 Nonionic detergent; used in blot detection procedures as a blocking reagent
or added to wash buffers to minimize nonspecific binding and background
Unstained standards Mixture of molecular weight marker proteins that do not have covalently attached
dye molecules; the bands are invisible during electrophoresis and transfer,
but are useful for molecular weight determination in stained gels
UreaChaotrope usually included at rather high concentrations (9.5 M) in sample
solubilization buffers for denaturing IEF and 2-D PAGE
Volt-hour (Vh)
Voltage multiplied by time is used as a unit for the duration of an IEF run
Prestained standards Mixture of molecular weight marker proteins that have covalently attached dye
molecules, which render the bands visible during electrophoresis and transfer
Western blotting Immobilization of proteins onto a membrane and subsequent detection by
protein-specific binding and detection reagents
RC DC™ assay kit
Zwitterion
Bio-Rad’s reductant- and detergent-compatible protein assay kit
Neutral molecule with positive and negative charges at different locations
ReadyStrip™ IPG strips Bio-Rad’s brand of IPG strips
ReadyPrep™ kits
128
Bio-Rad’s brand of sample preparation and 2-D electrophoresis kits and reagents
129
2-D Electrophoresis Guide
Appendices
Appendix B
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131
2-D Electrophoresis Guide
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Appendices
Related Bio-Rad Literature
Appendix C
Bio-Rad Laboratories, Inc. (2005) 2-D gel electrophoresis
troubleshooting. BioRadiations 116, 29–29.
Ordering Information
Tu C et al. (2010). Depletion of abundant plasma proteins and
limitations of plasma proteomics. J Proteome Res 9, 4982–4991.
Bulletin 1069 Colorimetric Protein Assays
Van den Bergh G and Arckens L (2008). Reducing sample complexity
by RP-HPLC: beyond the tip of the protein expression iceberg.
Methods Mol Biol 424, 147–156.
Bulletin 2587 High-Performance 2-D Gel Electrophoresis Using
Narrow pH-Range ReadyStrip IPG Strips
Bulletin 2414 The Little Book of Standards
Catalog # Description
Protein Sample Preparation Kits and Reagents
Wang W et al. (2008). Optimizing protein extraction from plant tissues
for enhanced proteomics analysis. J Sep Sci 31, 2032–2039.
Bulletin 2895 Protein Blotting Guide
Protein Extraction
163-2141
MicroRotofor ™ Cell Lysis Kit (Mammal), 15 preps,
includes 50 ml protein solubilization buffer (PSB),
ReadyPrep™ mini grinders (2 packs of 10 each)
Bulletin 2998 Protein Standards Application Guide
163-2142
Weiss W et al. (1992). Application of sequential extraction procedures
and glycoprotein blotting for the characterization of the 2-D
polypeptide patterns of barley seed proteins. Electrophoresis 13,
770–773.
Bulletin 3103 Removal of Abundant Myofilament Proteins from Rabbit
Myocardium Using the ReadyPrep Protein Extraction Kit (Membrane I)
Wessel D and Flugge UI (1984). A method for the quantitative recovery
of protein in dilute solution in the presence of detergents and lipids.
Anal Biochem 138, 141–143.
Bulletin 3110 Tips to Prevent Streaking on 2-D Gels
Bulletin 3131 The Rotofor System As a Prefractionation Device Used
Prior to Electrophoresis
Bulletin 3133 Molecular Weight Determination by SDS-PAGE
Westermeier R and Scheibe B (2008). Difference gel electrophoresis
based on lys/cys tagging. Methods Mol Biol 424, 73–85.
Bulletin 3144 Using Precision Plus Protein Standards to Determine
Molecular Weight
Wilkins MR et al. (1996). Progress with proteome projects: why all
proteins expressed by a genome should be identified and how to do it.
Biotechnol Genet Eng Rev 13, 19–50.
Bulletin 3145 Strategies for Protein Sample Preparation
Xixi E et al. (2006). Proteomic analysis of the mouse brain
following protein enrichment by preparative electrophoresis.
Electrophoresis 27, 1424–1431.
Bulletin 5344 Fractionation by Liquid-Phase Isoelectric Focusing in
the MicroRotofor Cell: Improved Detection of Low-Abundance Proteins
Yuan X and Desiderio DM (2005). Proteomics analysis of
prefractionated human lumbar cerebrospinal fluid. Proteomics 5,
541–550.
Zerefos PG et al. (2006). Characterization of the human urine
proteome by preparative electrophoresis in combination with 2-DE.
Proteomics 6, 4346–4355.
Zischka H et al. (2003). Improved proteome analysis of
Saccharomyces cerevisiae mitochondria by free-flow electrophoresis.
Proteomics 3, 906–916.
Zuo X and Speicher DW (2000). A method for global analysis of
complex proteomes using sample prefractionation by solution
isoelectrofocusing prior to two-dimensional electrophoresis.
Anal Biochem 284, 266–278.
Bulletin 5241 Important Factors Influencing Protein Solubility for
2-D Electrophoresis
Bulletin 5398 Enriching Basic and Acidic Rodent Brain Proteins with
Ion Exchange Spin Columns for Two-Dimensional Gel Electrophoresis
Bulletin 5754 Comparison of SYPRO Ruby and Flamingo Fluorescent
Gel Stains with Respect to Compatibility with Mass Spectrometry
Bulletin 5782 In-Gel Protein Quantitation Using the Criterion Stain Free
Gel Imaging System
Bulletin 5841 Quantitation of Serum and Plasma Proteins after
Enrichment of Low-Abundance Proteins with the ProteoMiner Protein
Enrichment System
Bulletin 5911 Mini-PROTEAN TGX Precast Gel: A Versatile and
Robust Laemmli-Like Precast Gel for SDS-PAGE
Bulletin 5939 Overcoming the Coomassie Blues
Bulletin 6040 A Guide to Polyacrylamide Gel Electrophoresis
and Detection
icroRotofor Cell Lysis Kit (Plant), 10 preps,
M
includes 50 ml protein solubilization buffer (PSB),
ReadyPrep 2-D cleanup kit (50 reaction size)
163-2143
MicroRotofor Cell Lysis Kit (Yeast), 15 preps,
includes 50 ml protein solubilization buffer (PSB),
15 ml yeast suspension buffer, 2 × 0.5 ml lyticase
(1.5 U/µl)
163-2144
MicroRotofor Cell Lysis Kit (Bacteria), 15 preps,
includes 50 ml protein solubilization buffer (PSB),
25 ml bacteria suspension buffer, 1 ml lysozyme
(1,500 U/µl)
Catalog # Description
Protein Fractionation
163-2100
ReadyPrep Sequential Extraction Kit, 5–15 preps
163-2085
ReadyPrep Protein Extraction Kit
(Soluble/Insoluble), 20 preps
163-2089
eadyPrep Protein Extraction Kit
R
(Cytoplasmic/Nuclear), 50 preps
163-2088
ReadyPrep Protein Extraction Kit (Membrane I),
50 preps
163-2084
ReadyPrep Protein Extraction Kit (Membrane II),
10 preps
163-2087
ReadyPrep Protein Extraction Kit (Signal),
50 preps
732-6711
Aurum™ CEX Mini Kit, 2 preps
732-6710
Aurum AEX Mini Kit, 2 preps
170-2800
MicroRotofor ™ Cell Kit, 100/120 V
163-2086
ReadyPrep™ Protein Extraction Kit (Total
Protein), 20 preps, general purpose protein
preparation kit, includes strong detergent ASB-14
170-2801
MicroRotofor Cell Kit, 220/240 V
170-2986
Rotofor® Purification System, 100/120 V
163-2146
ReadyPrep Mini Grinders, pkg of 20, 1.5 ml
grinding tube, contains grinding resin and matching
pestle, sufficient for twenty 100 mg extractions
170-2987
Rotofor Purification System, 220/240 V
170-2926
Model 491 Prep Cell, 100/120 V
170-2927
Model 491 Prep Cell, 220/240 V
163-2083
ReadyPrep 2-D Rehydration/Sample Buffer 1,
10 ml, protein solubilization reagent, includes 7 M
urea, 2 M thiourea, 1% ASB-14, 40 mM Tris, 0.001%
bromophenol blue
170-2908
Mini Prep Cell without Reagent Starter Kit
163-2106
ReadyPrep 2-D Starter Kit Rehydration/Sample
Buffer, 10 ml, protein sample buffer, includes 8
M urea, 2% CHAPS, 50 mM DTT, 0.2% Bio-Lyte®
ampholyte, 0.001% bromophenol blue
732-6712
163-3006
ProteoMiner ™ Protein Enrichment SmallCapacity Kit, 10 preps for 10 mg total protein
163-2145
Protein Solubilization Buffer (PSB), pkg of 1,
strongly chaotropic protein solubilization buffer,
contains NDSB 201, urea, thiourea, and CHAPS,
makes 50 ml solution
Protein Assay Kits and Instruments
Protein Sample Depletion
732-6701
Aurum Serum Protein Mini Kit, 10 preps
Aurum™ Affi-Gel® Blue Mini Kit, 2 preps
163-3007
ProteoMiner Protein Enrichment LargeCapacity Kit, 10 preps for 50 mg total protein
500-0001
Bio-Rad Protein Assay Kit I, includes 450 ml dye
reagent concentrate, bovine g-globulin standard;
sufficient for 440 standard assays or 2,200
microplate assays
163-2091
ReadyPrep Proteomics Grade Water, 500 ml
161-0730 Urea, 250 g
161-0719 Tris, 1 kg
161-0460
CHAPS, 1g
Bulletin 6139 Versatile Separation Capabilities of the
PROTEAN i12 IEF System
161-0611
Dithiothreitol (DTT), 5 g
500-0002
Bio-Rad Protein Assay Kit II, includes 450 ml
dye reagent concentrate, bovine serum albumin
standard; sufficient for 440 standard assays or
2,200 microplate assays
Bulletin 6140 Use of the PROTEAN i12 IEF System for In-Gel
Peptide Fractionation Prior to LC-MS and Comparison with
Off-Gel Fractionation
163-2101
Tributylphosphine (TBP), 0.6 ml, 200 mM
500-0111
163-2109
Iodoacetamide, 30 g
161-0404 Bromophenol Blue, 10 g
DC™ Protein Assay Kit I, includes 250 ml alkaline
copper tartrate solution, 2 L dilute Folin reagent,
5 ml surfactant solution, bovine g-globulin standard;
sufficient for 450 standard assays
500-0112
C Protein Assay Kit II, includes 250 ml alkaline
D
copper tartrate solution, 2 L dilute Folin reagent, 5 ml
surfactant solution, bovine serum albumin standard;
sufficient for 450 standard assays
500-0120
RC DC™ Protein Assay Reagents Package,
includes RC reagents package and DC reagents
package, and sufficient for 450 standard assays
Bulletin 6138 PROTEAN i12 IEF System: Independent Voltage
and Current Control Enables Optimization of First-Dimension
IEF Conditions
Bulletin 4006173 Instruction Manual, SYPRO Ruby Protein Stains
Bulletin 4307051 Instruction Manual, Bio-Safe Coomassie Stain
Protein Sample Cleanup
163-2130
ReadyPrep 2-D Cleanup Kit, 50 preps
Bulletin 10003321 Instruction Manual, Flamingo Fluorescent Gel Stain
163-2140
ReadyPrep 2-D Cleanup Kit, 5 preps
Bulletin 10017295 Instruction Manual, Oriole Fluorescent Gel Stain
732-6221
Micro Bio-Spin™ 6 Columns, includes 25 columns
in Tris buffer, 50 collection tubes
Bulletin 10022069 Instruction Manual, PROTEAN i12 IEF System
Bulletin LIT-442 Instruction Manual, Silver-Stain Plus.
732-6227
Bio-Spin® 6 Columns, includes 25 columns in
Tris buffer, 50 collection tubes
732-6228
io-Spin 6 Columns, includes 100 columns in
B
Tris buffer, 200 collection tubes
163-2090
ReadyPrep Reduction-Alkylation Kit, 100 preps
500-0121
RC DC Protein Assay Kit I, includes RC reagents
package, DC reagents package, bovine g-globulin
standard; sufficient for 450 standard assays
500-0122
RC DC Protein Assay Kit II, includes RC reagents
package, DC reagents package, bovine serum
albumin standard; sufficient for 450 standard assays
500-0201
Quick Start™ Bradford Protein Assay Kit 1,
includes 1× dye reagent (1 L), bovine serum albumin
standard (5 × 2 mg/ml); sufficient for 200 standard
assays or 4,000 microplate assays
132
133
2-D Electrophoresis Guide
Appendices
Catalog # Description
Catalog # Description
Catalog # 500-0202
Quick Start Bradford Protein Assay Kit 2,
includes 1× dye reagent (1 L), bovine serum albumin
standard set (2 sets of 7 concentration standards,
0.125–2.0 mg/ml, 2 ml)
161-0395
Precision Plus Protein Kaleidoscope Standards
Value Pack, 250 applications
500-0203
Quick Start Bradford Protein Assay Kit 3,
includes 1× dye reagent (1 L), bovine g-globulin
standard (5 × 2 mg/ml)
161-0397
recision Plus Protein Dual Xtra Standards
P
Value Pack, 250 applications
161-0385 Precision Plus Protein™ WesternC™ Pack,
50 applications
500-0204
Quick Start Bradford Protein Assay Kit 4,
includes 1× dye reagent (1 L), bovine g-globulin
standard set (2 sets of 7 concentration standards,
0.125–2.0 mg/ml, 2 ml)
170-2525
SmartSpec™ Plus Spectrophotometer
170-2502
Standard Cuvette, 1–3.5 ml, quartz
170-2511
trUView™ Cuvettes, pack of 100, individually
packaged, disposable DNase- and
RNase-free cuvettes
Protein Standards
™
161-0378
Precision Plus Protein Standard Plugs,
pkg of 24, 1 mm thick agarose plugs containing
10 Strep-tagged recombinant proteins (10–250 kD),
including three reference bands
161-0363 Precision Plus Protein Unstained Standards,
100 applications
161-0396
Precision Plus Protein Unstained Standards
Value Pack, 500 applications
161-0377 Precision Plus Protein Dual Xtra Standards,
50 applications
161-0398
Precision Plus Protein WesternC Standards Pack
Value Pack, 250 applications
161-0399
recision Plus Protein WesternC Standards
P
Value Pack, 250 applications
161-0324 Kaleidoscope™ Prestained Standards,
broad range, 500 μl
161-0325 Kaleidoscope Polypeptide Standards, 500 μl
161-0309 Prestained SDS-PAGE Standards, high range,
500 μl
161-0305 Prestained SDS-PAGE Standards, low range,
500 μl
161-0304 SDS-PAGE Standards, low range, 200 μl
161-0317 SDS-PAGE Standards, broad range, 200 μl
165-4035 7 cm i12 Rehydration/Equilibration Tray,
with lids, pkg of 25
recision Plus Protein Dual Color Standards
P
Value Pack, 250 applications
163-2096 ReadyStrip 100× pH 5.5–6.7 Buffer, includes only
ampholytes, 1 ml
ReadyStrip 100× pH 7–10 Buffer, includes only
ampholytes, 1 ml
163-2095 ReadyStrip 100× pH 6.3–8.3 Buffer, includes only
ampholytes, 1 ml
Ampholyte
pH Range
6/8
5/7
5/8
7/9
163-1112
163-1132
163-1142
163-1162
163-1152
163-1192
163-1172
163-1182
25 ml
163-1113
—163-1143
163-1163
163-1153
163-1193
——
IPG Strips and Buffers
ReadyStrip IPG strips, 12 per package.
134
165-4025 11 cm i12 Rehydration/Equilibration Tray,
with lids, pkg of 25
164-6313 13 cm i12 Rehydration/Equilibration Tray,
with lids, pkg of 25
165-4015 17 cm i12 Rehydration/Equilibration Tray,
with lids, pkg of 25
165-4041 18 cm i12 Rehydration/Equilibration Tray,
with lids, pkg of 25
165-4043 24 cm i12 Rehydration/Equilibration Tray,
with lids, pkg of 25
164-6040 IPG Strip Retainers, pkg of 2
164-6020 i12 Sample Cup Holder, pkg of 1, 12-position
sample cup holder, includes 25 disposable
sample cups
164-6021 i12 Sample Cups, pkg of 25
164-6030 Gel-Side Up Electrode Wicks, pkg of 100
164-6031 Gel-Side Down Electrode Wicks, pkg of 500
8/10
1 ml
163-2094
———
————
10 ml
13 cm i12 Focusing Tray, includes 2 IPG strip retainers
17 cm i12 Focusing Tray, includes 2 IPG strip retainers
18 cm i12 Focusing Tray, includes 2 IPG strip retainers
163-2097 ReadyStrip 100× pH 4.7–5.9 Buffer, includes only
ampholytes, 1 ml
4/6
164-6113 164-6117 24 cm i12 Focusing Tray, includes 2 IPG strip retainers
recision Plus Protein Dual Color Standards,
P
50 applications
3/5
11 cm i12 Focusing Tray, includes 2 IPG strip retainers
164-6118 161-0374 3/10
164-6111 164-6124 163-2098 ReadyStrip 100× pH 3.9–5.1 Buffer, includes only
ampholytes, 1 ml
7 cm i12™ Focusing Tray, includes 2 IPG strip retainers
SDS-PAGE Standards, high range, 200 μl
161-0393
Precision Plus Protein All Blue Standards Value
Pack, 500 applications
Bio-Lyte ®
164-6107 161-0303 ™
Catalog # 164-6012 Negative Electrode Assembly, pkg of 1
164-6011 Positive Electrode Assembly, pkg of 1
Description
Mini-PROTEAN ® Tetra Cells and Systems
165-8000 Mini-PROTEAN Tetra Cell, 10-well, 0.75 mm
thickness; 4-gel system includes 5 combs, 5 sets
of glass plates, 2 casting stands, 4 casting frames,
sample loading guide, electrode assembly,
companion running module, tank, lid with power
cables, mini cell buffer dam
165-8001
Mini-PROTEAN Tetra Cell, 10-well, 1.0 mm
thickness; 4-gel system includes 5 combs, 5 sets
of glass plates, 2 casting stands, 4 casting frames,
sample loading guide, electrode assembly,
companion running module, tank, lid with power
cables, mini cell buffer dam
165-8002
ini-PROTEAN Tetra Cell, 10-well, 0.75 mm
M
thickness; 2-gel system includes 5 combs, 5 sets of
glass plates, casting stand, 2 casting frames, sample
loading guide, electrode assembly, tank, lid with
power cables, mini cell buffer dam
165-8003
ini-PROTEAN Tetra Cell, 10-well, 1.0 mm
M
thickness; 2-gel system includes 5 combs, 5 sets of
glass plates, casting stand, 2 casting frames, sample
loading guide, electrode assembly, tank, lid with
power cables, mini cell buffer dam
164-6001 PROTEAN i12 IEF Cell, 90–240 VAC basic
unit includes cell, positive and negative
electrode assemblies
restained SDS-PAGE Standards, broad range,
P
500 μl
163-2093 161-0375 Precision Plus Protein™ Kaleidoscope™
Standards, 50 applications
PROTEAN ® i12™ IEF System
164-6000 PROTEAN i12 IEF System, 90–240 VAC, includes
basic unit, positive and negative electrode
assemblies, 7 cm, 11 cm, and 17 cm focusing trays
with IPG strip retainers, 1 pack each of 7 cm, 11 cm,
and 17 cm rehydration/equilibration trays, 2 pairs of
forceps, 2 packs electrode wicks for gel-side down
and gel-side up applications, mineral oil, 2 cleaning
brushes, cleaning concentrate, 2 USB flash drives,
3 styluses, pH 3–10 ReadyStrip™ IPG strips in 7 cm,
11 cm, and 17 cm lengths, rehydration sample
buffer, and instruction manual. 13 cm, 18 cm,
and 24 cm trays and cup loading accessories
can be purchased separately
161-0318 161-0373 Precision Plus Protein All Blue Standards,
50 applications
161-0394
Description
Electrophoresis Instrumentation
165-8004 Mini-PROTEAN Tetra Cell for Mini Precast Gels,
4-gel system includes electrode assembly, clamping
frame, companion module, tank, lid with power
cables, mini cell buffer dam
165-8005
ini-PROTEAN Tetra Cell for Mini Precast Gels,
M
2-gel system includes electrode assembly,
clamping frame, tank, lid with power cables,
mini cell buffer dam
165-8006 Mini-PROTEAN Tetra Cell, 10-well, 1.5 mm
thickness; 4-gel system includes 5 combs, 5 sets
of glass plates, 2 casting stands, 4 casting frames,
sample loading guide, electrode assembly,
companion running module, tank, lid with power
cables, mini cell buffer dam
165-8007
Mini-PROTEAN Tetra Cell, 10-well, 1.5 mm
thickness; 2-gel system includes 5 combs, 5 sets of
glass plates, casting stand, 2 casting frames, sample
loading guide, electrode assembly, tank, lid with
power cables, mini cell buffer dam
165-8025 Mini-PROTEAN Tetra Cell and PowerPac™ Basic
Power Supply, includes 165-8001 and 164-5050
165-8026 Mini-PROTEAN Tetra Cell and PowerPac™
Universal Power Supply, includes 165-8001
and 164-5070
165-8027 ini-PROTEAN Tetra Cell and PowerPac™ HC
M
Power Supply, includes 165-8001 and 164-5052
ini-PROTEAN Tetra Cell and PowerPac™ HV
M
Power Supply, includes 165-8001 and 164-5056
7 cm
11 cm
17 cm
18 cm
24 cm
164-6010 Electrode Assembly Pair, pkg of 1 pair, positive
and one negative electrode assemblies
165-8028 pH 3–10
163-2000
163-2014
163-2007
163-2032
163-2042
165-4072 Cleaning Brushes, pkg of 2
161-0722 Cleaning Concentrate
165-8029 Mini-PROTEAN Tetra Cell and Mini Trans-Blot®
Module, includes 165-8001 and 170-3935
pH 3–10 NL
163-2002
163-2016
163-2009
163-2033
163-2043
pH 3–6
163-2003
163-2017
163-2010
163-2035
163-2045
164-6060 USB Flash Drive, pkg of 2
Stylus, pkg of 3
pH 4–7
163-2001
163-2015
163-2008
163-2034
163-2044
164-6050 pH 5–8
163-2004
163-2018
163-2011
163-2036
163-2046
165-4070 Forceps, pkg of 1
Mineral Oil
ReadyPrep 2-D Starter Kit
pH 7–10
163-2005
163-2019
163-2012
163-2037
163-2047
163-2129 pH 3.9–5.1
163-2028
163-2024
163-2020
163-2038
163-2048
163-2105
pH 4.7–5.9
163-2029
163-2025
163-2021
163-2039
163-2049
pH 5.5–6.7
163-2030
163-2026
163-2022
163-2040
163-2050
pH 6.3–8.3
163-2031
163-2027
163-2023
163-2041
163-2051
165-8030 Mini-PROTEAN Tetra Cell for Ready Gel Precast
Gels and Mini Trans-Blot Module, includes
165-8004 and 170-3935
165-8033 Mini-PROTEAN Tetra Cell, Mini Trans-Blot
Module, and PowerPac Basic Power Supply,
includes 165-8001, 170-3935, and 164-5050
135
2-D Electrophoresis Guide
Appendices
Catalog # Description
Mini-PROTEAN ® Tetra Cells and Systems (contd.)
165-8034 Mini-PROTEAN Tetra Cell for Ready Gel Precast
Gels, Mini Trans-Blot Module, and PowerPac
Basic Power Supply, includes 165-8004, 170-3935,
and 164-5050
165-8035 Mini-PROTEAN Tetra Cell, Mini Trans-Blot
Module, and PowerPac HC Power Supply,
includes 165-8001, 170-3935, and 164-5052
165-8036 Mini-PROTEAN Tetra Cell for Ready Gel Precast
Gels, Mini Trans-Blot Module, and PowerPac HC
Power Supply, includes 165-8004, 170-3935,
and 164-5052
Mini-PROTEAN Dodeca Cells and Systems
165-4100
Mini-PROTEAN ® 3 Dodeca™ Cell, includes
electrophoresis tank with built-in cooling coil, lid with
power cables, 6 electrophoresis clamping frames,
2 buffer dams, drain line, 2 gel releasers
165-4101
Mini-PROTEAN 3 Dodeca Cell with Multi-Casting
Chamber, same as 165-4100 with multi-casting
chamber, 15 separation sheets, 8 acrylic blocks,
tapered luer connector, stopcock valve
™
Criterion Cells and Systems
165-6001
Criterion Cell, includes buffer tank, lid with power
cables, 3 sample loading guides (12 + 2-well,
18-well, 26-well)
Catalog # Description
Catalog # 165-3190
ROTEAN II XL Cell, wide format 1-D vertical
P
electrophoresis cell, 2.0 mm, includes PROTEAN II
xi basic unit (#165-1834) and 2.0 mm IPG conversion
kit (#165-3184)
PROTEAN ® Plus Dodeca™ Cells and Systems (contd.)
165-5134 PROTEAN Plus Dodeca Cell (100/120 V) and
Two 6-Row AnyGel Stands, includes 165-4150
and two 165-5131
165-1815
PROTEAN II xi Cell 2-D Conversion Kit,
converts PROTEAN II xi cell into a tube gel IEF
2-D system; includes 2 tube gel adaptors, 24
glass tubes (1.5 mm ID, 180 mm length), gaskets,
grommets, stoppers
165-3183
PROTEAN II xi Cell IPG Conversion Kit, 1.0 mm,
18.5 × 20 cm, for conversion to IPG PROTEAN II
XL system; includes IPG clamps, 20 × 20 cm glass
plates (2), IPG spacers, 2-D combs, and central
cooling core gaskets
165-3186
PROTEAN II xi Cell IPG Conversion Kit, 1.5 mm,
18.5 × 20 cm, for conversion to IPG PROTEAN II
XL system; includes IPG clamps, 20 × 20 cm glass
plates (2), IPG spacers, 2-D combs, and central
cooling core gaskets
165-5133 Criterion Dodeca Cell and 6-Row AnyGel™ Stand,
includes 165-4130 and 165-5131
PROTEAN ® II xi Cells
165-1801 PROTEAN II xi Cell, 16 cm, without spacers
and combs
165-1802 PROTEAN II xi Cell, 16 cm, 1.5 mm spacers (4),
15-well combs (2)
165-1803 165-1804 ROTEAN II xi Cell, 16 cm, 1.0 mm spacers (4),
P
15-well combs (2)
ROTEAN II xi Cell, 16 cm, 0.75 mm spacers (4),
P
15-well combs (2)
165-1811 PROTEAN II xi Cell, 20 cm, without spacers
and combs
165-1812 PROTEAN II xi Cell, 20 cm, 1.5 mm spacers (4),
15-well combs (2)
165-1813 PROTEAN II xi Cell, 20 cm, 1.0 mm spacers (4),
15-well combs (2)
165-1814 ROTEAN II xi Cell, 20 cm, 0.75 mm spacers (4),
P
15-well combs (2)
PROTEAN II XL Cells
165-3188
PROTEAN II XL Cell, wide format 1-D vertical
electrophoresis cell, 1.0 mm, includes PROTEAN II
xi basic unit (#165-1834) and 1.0 mm IPG conversion
kit (#165-3183)
165-3189
PROTEAN II XL Cell, wide format 1-D vertical
electrophoresis cell, 1.5 mm, includes PROTEAN II
xi basic unit (#165-1834) and 1.5 mm IPG conversion
kit (#165-3186)
136
10% Criterion Tris-HCl Precast Gel
12.5% Criterion Tris-HCl Precast Gel
345-0103
4–15% Criterion Tris-HCl Precast Gel
345-0104
4–20% Criterion Tris-HCl Precast Gel
345-0105
8–16% Criterion Tris-HCl Precast Gel
345-0106
10.5–14% Criterion Tris-HCl Precast Gel
345-0107
10–20% Criterion Tris-HCl Precast Gel
345-0115
10% Criterion XT Bis-Tris Precast Gel
345-0121
12% Criterion XT Bis-Tris Precast Gel
345-0127
4–12% Criterion XT Bis-Tris Precast Gel
345-0133
3–8% Criterion XT Tris-Acetate Precast Gel
345-8161
8–16% Criterion Stain-Free Precast Gel
165-4145 165-5135 ROTEAN Plus Dodeca Cell (220/240 V)
P
Trans-Blot Plus Cell, and PowerPac Universal
Power Supply, includes 165-4151, 170-3990,
and 164-5070
ROTEAN Plus Dodeca Cell (220/240 V) and
P
Two 6-Row AnyGel Stands, includes 165-4151
and two 165-5131
PowerPac HV Power Supply, 100–120/220–240 V
164-5070 owerPac Universal Power Supply,
P
100–120/220–240 V
165-1956
165-3176
ROTEAN II xi Multi-Cell 2-D Conversion Kit,
P
for proper cooling in 2-D electrophoresis
applications; includes 2 cooling coils and manifold
ROTEAN II XL Multi-Cell, wide format vertical
P
electrophoresis multi-cell, 1.0 mm, compatible with
ReadyStrip IPG strips; includes catalog #165-1951,
#165-1956, and 3 PROTEAN II xi cell IPG conversion
kits of desired thickness
165-3177
PROTEAN II XL Multi-Cell, wide format vertical
electrophoresis multi-cell, 1.5 mm, compatible with
ReadyStrip IPG strips; includes catalog #165-1951,
#165-1956, and 3 PROTEAN II xi cell IPG conversion
kits of desired thickness
165-3178
PROTEAN II XL Multi-Cell, wide format vertical
electrophoresis multi-cell, 2.0 mm, compatible with
ReadyStrip IPG strips; includes catalog #165-1951,
#165-1956, and 3 PROTEAN II xi cell IPG conversion
kits of desired thickness
PROTEAN ® Plus Dodeca™ Cells and Systems
165-4150 PROTEAN Plus Dodeca Cell, 100/120 V, includes
electrophoresis buffer tank with built-in ceramic
cooling core, lid, buffer recirculation pump with
tubing, 2 gel releasers
165-4140 PROTEAN Plus Dodeca Cell (100/120 V) and
PowerPac HC Power Supply, includes 165-4150
and 164-5052
165-4142 PROTEAN Plus Dodeca Cell (100/120 V) and
PowerPac Universal Power Supply, includes
165-4150 and 164-5070
165-4144 PROTEAN Plus Dodeca Cell (100/120 V),
Trans-Blot Plus Cell, and PowerPac Universal
Power Supply, includes 165-4150, 170-3990,
and 164-5070
12% Mini-PROTEAN TGX Stain-Free Precast Gel
456-8121
A ny kD Mini-PROTEAN TGX Stain-Free
Precast Gel
345-0102
164-5056 165-4138 10% Mini-PROTEAN TGX Stain-Free Precast Gel
456-8041
345-0101
PowerPac HC Power Supply, 100–120/220–240 V
ROTEAN II xi Multi-Cell, multi-cell electrophoresis
P
system, includes 3 central cooling cores, buffer
tank, PROTEAN II xi multi-casting chamber
with accessories
456-8031
165-4143 PROTEAN Plus Dodeca Cell (220/240 V) and
PowerPac Universal Power Supply, includes
165-4151 and 164-5070
164-5052
165-1951
Description
Mini-PROTEAN ® TGX Stain-Free™ Precast Gels
(for 7 cm IPG Strips)
456-8021
7.5% Mini-PROTEAN TGX Stain-Free Precast Gel
Criterion Precast Gels
(for 11 cm IPG Strips)
IPG +1 well, package of 1
165-1834
ROTEAN II xi Basic Unit With Casting
P
Stand, vertical electrophoresis system, includes
electrophoresis cell with central cooling core, gel
casting stand
Catalog # 165-4141 PROTEAN Plus Dodeca Cell (220/240 V) and
PowerPac HC Power Supply, includes 165-4151
and 164-5052
Power Supplies
164-5050
PowerPac™ Basic Power Supply,
100–120/220–240 V
Criterion™ Dodeca™ Cells and Systems
165-4130 Criterion Dodeca Cell, includes electrophoresis
buffer tank with built-in cooling coil, lid with
power cables
165-4139 Criterion Dodeca Cell and PowerPac Universal
Power Supply, includes 165-4130 and 164-5070
ROTEAN Plus Dodeca Cell, 220/240 V, includes
P
electrophoresis buffer tank with built-in ceramic
cooling core, lid, buffer recirculation pump with
tubing, 2 gel releasers
165-3184
PROTEAN II xi Cell IPG Conversion Kit, 2.0 mm,
18.5 × 20 cm, for conversion to IPG PROTEAN II
XL system; includes IPG clamps, 20 × 20 cm glass
plates (2), IPG spacers, 2-D combs, and central
cooling core gaskets
165-6019
Criterion Cell and PowerPac Basic Power Supply,
100–120/220–240 V, includes 165-6001 and
164-5050
riterion Dodeca Cell and PowerPac HC Power
C
Supply, includes 165-4130 and 164-5052
165-4151 Description
Criterion™ TGX™ Precast Gels
(for 11 cm IPG Strips)
567-1071
18% Criterion TGX Precast Gel
IEF and SDS-PAGE Buffers and Reagents
567-1081
4–15% Criterion TGX Precast Gel
163-2111
ReadyPrep Overlay Agarose, 1 bottle, 50 ml
567-1091
4–20% Criterion TGX Precast Gel
161-0732 10× Tris/Glycine/SDS, 1 L
567-1101
8–16% Criterion TGX Precast Gel
161-0734 10× Tris/Glycine, 1 L
567-1111
10–20% Criterion TGX Precast Gel
161-0744
10× Tris/Tricine/SDS, 1 L
161-0788 XT MOPS Running Buffer, 20×, 500 ml
567-1121
Any kD Criterion TGX Precast Gel
161-0789 XT MES Running Buffer, 20×, 500 ml
161-0790 XT Tricine Running Buffer, 20×, 500 ml
161-0793 T MOPS Buffer Kit, includes 500 ml 20× XT
X
MOPS running buffer, 10 ml 4× XT sample buffer,
1 ml 20× XT reducing agent
161-0796 T MES Buffer Kit, includes 500 ml 20× XT MES
X
running buffer, 10 ml 4× XT sample buffer, 1 ml
20× XT reducing agent
161-0797 XT Tricine Buffer Kit, includes 500 ml 20× XT
Tricine running buffer, 10 ml 4× XT sample buffer,
1 ml 20× XT reducing agent
Criterion™ TGX Stain-Free™ Precast Gels
(for 11 cm IPG Strips)
567-8071
18% Criterion TGX Stain-Free Precast Gel
567-8081
4–15% Criterion TGX Stain-Free Precast Gel
567-8091
4–20% Criterion TGX Stain-Free Precast Gel
567-8101
8–16% Criterion TGX Stain-Free Precast Gel
567-8111
10–20% Criterion TGX Stain-Free Precast Gel
567-8121
Any kD Criterion TGX Stain-Free Precast Gel
* For a complete selection of precast gels, visit www.bio-rad.com
Gel Casting Buffers and Reagents
161-0729 EDTA, 500 g
161-0718 Glycine, 1 kg
161-0713 Tricine, 500 g
161-5100
SDS-PAGE Reagent Starter Kit, includes
100 g acrylamide, 5 g bis, 5 ml TEMED,
10 g ammonium persulfate
161-0719 Tris, 1 kg
161-0100
Acrylamide, 99.9%, 100 g
161-0404
Bromophenol Blue, 10 g
161-0120
Acrylamide/Bis Powder, 19:1, 30 g
Precast Gels*
161-0122
Acrylamide/Bis Powder, 37.5:1, 30 g
Mini-PROTEAN ® TGX™ Precast Gels
(for 7 cm IPG Strips)
IPG well, 10 gels per box
161-0140
40% Acrylamide Solution, 500 ml
161-0144
40% Acrylamide/Bis Solution, 19:1, 500 ml
161-0146
40% Acrylamide/Bis Solution, 29:1, 500 ml
161-0148
40% Acrylamide/Bis Solution, 37.5:1, 500 ml
161-0154
30% Acrylamide/Bis Solution, 19:1, 500 ml
161-0156
30% Acrylamide/Bis Solution, 29:1, 500 ml
161-0158
30% Acrylamide/Bis Solution, 37.5:1, 500 ml
161-0200
Bis Crosslinker, 5 g
456-1021
7.5% Mini-PROTEAN TGX Precast Gel
456-1031
10% Mini-PROTEAN TGX Precast Gel
456-1041
12% Mini-PROTEAN TGX Precast Gel
456-1081
4–15% Mini-PROTEAN TGX Precast Gel
456-1091
4–20% Mini-PROTEAN TGX Precast Gel
456-9031
Any kD™ Mini-PROTEAN TGX Precast Gel
137
2-D Electrophoresis Guide
Catalog # Appendices
Description
Catalog # Description
Benzonase is a trademark of Merck KGaA Corporation.
Gel Casting Buffers and Reagents (contd.)
High-Throughput Stainers
Coomassie is a trademark of BASF Aktiengesellschaft.
161-0800
TEMED, 5 ml
165-3400
Cy is a trademark of GE Healthcare Group Companies.
161-0798
Resolving Gel Buffer, 1.5 M Tris-HCl, pH 8.8, 1 L
161-0700
Ammonium Persulfate (APS), 10 g
Dodeca Stainer, large, 100–240 V, includes
13 trays (12 clear, 1 white), 12 tray attachments,
shaking rack, solution tank, lid with shaker motor,
shaker control unit, gel clip
161-0799
Stacking Gel Buffer, 0.5 M Tris-HCl, pH 6.8, 1 L
Dodeca Stainer, small, 100–240 V, includes
13 trays (12 clear, 1 white), 12 Criterion tray
attachments, shaking rack, solution tank, lid with
shaker motor, shaker control unit, gel clip
Triton is a trademark of Dow Chemical Company.
165-3401
Gel Casting Accessories
See catalog or www.bio-rad.com for a complete listing of
accessories, including available empty gel cassettes and glass
plates, spacers, combs, etc.
165-5131 AnyGel™ Stand, 6-row, holds 6 PROTEAN gels,
12 Criterion gels, or 18 Ready Gel mini gels
165-4131 Imaging Systems and Spot Cutter
GS-900™ Calibrated Densitometry System, gel
densitometry system, PC compatible, scanner,
cables, Image Lab software, optional 21 CFR Part 11
and Instrument Qualification/Operations Qualification
170-8280 ChemiDoc™ MP System, gel imaging system,
PC or Mac, includes darkroom, UV transilluminator,
epi-white illumination, camera, power supply, cables,
Image Lab™ software
Gel Doc EZ System, gel imaging system, PC or Mac, includes darkroom, camera, cables, Image Lab software; samples trays (#170-8271, 170-8272, 170-8273, or 170-8274) are sold separately; sample trays are required to use the system
Molecular Imager® PharosFX™ Plus System,
PC or Mac, 110–240 V, includes Quantity One®
software, sample tray set, fluorescence filters
(170-7866, 170-7896) and phosphor imaging filters,
USB2 cable
A nyGel Stand, single-row, holds 1 PROTEAN gel,
2 Criterion gels, or 3 Ready Gel mini gels
165-4122
Model 485 Gradient Former and Mini-PROTEAN
3 Multi-Casting Chamber, includes 165-4120
and 165-4110
165-4123
Model 495 Gradient Former and PROTEAN Plus
Multi-Casting Chamber, includes 165-4121
and 165-4160
161-0786 Bio-Safe™ Coomassie Stain, 1 L
170-8270
161-0787 Bio-Safe Coomassie Stain, 5 L
170-9460
Total Protein Gel Stains
161-0449 Silver Stain Plus™ Kit, includes fixative enhancer
concentrate, silver complex solution, reduction
moderator solution, image development reagent,
development accelerator reagent, stains 13 full size
or 40 mini gels
TABLE OF CONTENTS
170-7991 161-0496 Oriole™ Fluorescent Gel Stain, 1× solution, 1 L
161-0492 Flamingo™ Fluorescent Gel Stain, 10× solution,
500 ml
170-3125 SYPRO Ruby Protein Gel Stain, 1× solution, 1 L
161-0440 inc Stain and Destain Kit, includes 125 ml of 10×
Z
zinc stain solution A, 125 ml of 10× zinc stain solution
B, 125 ml of 10× zinc destain solution
Parafilm is a trademark of American National Can Company.
Pro-Q, Qdot, and SYPRO are trademarks of
Invitrogen Corporation.
Tween is a trademark of ICI Americas Inc.
Tygon is a trademark of Norton Company.
Whatman is a trademark of Whatman Limited Corporation.
ZipTip is a trademark of Millipore Corporation.
Bio-Rad Laboratories, Inc. is licensed by Invitrogen
Corporation to sell SYPRO products for research use only
under U.S. Patent Number 5,616,502.
Precision Plus Protein standards are sold under license from
Life Technologies Corporation, Carlsbad, CA, for use only by
the buyer of the product. The buyer is not authorized to sell
or resell this product or its components.
Purchase of Criterion XT Bis-Tris gels, XT MOPS running
buffer, XT MES running buffer, XT MOPS buffer kit, and
XT MES buffer kit is accompanied by a limited license
under U.S. Patent Numbers 6,143,154; 6,096,182; 6,059,948;
5,578,180; 5,922,185; 6,162,338; and 6,783,651, and
corresponding foreign patents.
StrepTactin is covered by German patent application
P 19641876.3. Bio-Rad Laboratories, Inc. is licensed by
Institut fur Bioanalytik GmbH to sell these products for
research use only.
170-9450
Molecular Imager PharosFX System, PC or Mac,
110–240 V, includes Quantity One software, sample
tray set, fluorescence filters (170-7866, 170-7896),
USB2 cable
165-7200
E XQuest™ Spot Cutter, gel excision instrument,
includes enclosure, imaging system, fluidics system,
robotics, sensors, cutting head, gel tray, microplate
rack, wash station
165-7201
161-0470 Copper Stain and Destain Kit, includes 125 ml
of 10× copper stain, 125 ml of 10× copper
destain solution
XQuest Spot Cutter with PC, gel excision
E
instrument, includes PC, enclosure, imaging system,
fluidics system, robotics, sensors, cutting head,
gel tray, microplate rack, wash station
Analysis Software
170-9690
Image Lab Software
170-9600
Quantity One 1-D Analysis Software, PC or Mac
170-9630
PDQuest™ Advanced 2-D Analysis Software
Gel Drying Supplies
165-1771
GelAir ™ Drying System, 115 V, 60 Hz, includes
165-1777, 2 drying frames, 16 clamps, assembly
table, 50 precut sheets of cellophane support,
gel drying solution
165-1777
138
GelAir Dryer, 115 V, 60 Hz, gel drying oven only
139
Bio-Rad
Laboratories, Inc.
Web site www.bio-rad.com USA 800 424 6723 Australia 61 2 9914 2800 Austria 01 877 89 01 Belgium 09 385 55 11 Brazil 55 11 5044 5699
Canada 905 364 3435 China 86 21 6169 8500 Czech Republic 420 241 430 532 Denmark 44 52 10 00 Finland 09 804 22 00
France 01 47 95 69 65 Germany 089 31 884 0 Greece 30 210 9532 220 Hong Kong 852 2789 3300 Hungary 36 1 459 6100 India 91 124 4029300
Israel 03 963 6050 Italy 39 02 216091 Japan 03 6361 7000 Korea 82 2 3473 4460 Mexico 52 555 488 7670 The Netherlands 0318 540666
New Zealand 64 9 415 2280 Norway 23 38 41 30 Poland 48 22 331 99 99 Portugal 351 21 472 7700 Russia 7 495 721 14 04
Singapore 65 6415 3188 South Africa 27 861 246 723 Spain 34 91 590 5200 Sweden 08 555 12700 Switzerland 026 674 55 05
Taiwan 886 2 2578 7189 Thailand 800 88 22 88 United Kingdom 020 8328 2000
Life Science
Group
Bulletin 2651 Rev F
US/EG
13-0893
0413
Sig 1212
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