Bio-Rad GS-900™ Calibrated Densitometer - Manual (Page 2)

2-D Electrophoresis Guide
1
Table of Contents
Part I: Theory and Product Selection
5
Chapter 1 Overview of
Two-Dimensional Electrophoresis
5
The Context of Proteomics 6
Overview of Experimental Design
6
Sample Preparation 8
First-Dimension Separation: IEF 8
Second-Dimension Separation: SDS-PAGE 8
Detection 9
Image Acquisition, Analysis, and Spot Cutting 9
Protein Digestion and Identification by Mass Spectrometry 9
Chapter 2 Sample Preparation
11
The Importance of Sample Preparation
12
General Considerations
12
Cell Lysis 12
Protein Solubilization
15
Chaotropic Agents 15
Detergents 15
Reducing Agents 16
Ampholytes, Buffers, and Other Additives 17
Removal of Interfering Substances 19
General Considerations 19
Nucleic acids (DNA and RNA) 19
Polysaccharides 20
Phenolic Compounds 20
Lipids 20
Salts and Other Small Ionic Compounds 20
Prevention of Keratin Contamination 21
Prefractionation
22
Fractionation by Subcellular Location 22
Fractionation by Solubility/Hydrophobicity 24
Fractionation by Protein Charge 24
Fractionation by pI 25
Fractionation by Size (MW) 26
Depletion and Dynamic Range Reduction
27
Depletion 27
Dynamic Range Reduction 28
ProteoMiner Technology 29
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
36
Choice of pH Gradient 36
Choice of IPG Strip Length 36
Estimation of pl 37
Sample Application
38
Sample Application during Rehydration 38
Sample Application by Cup Loading 39
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
44
Choice of Gel Percentage (Composition) 45
Choice of Gel Size 47
Choice of Buffer System 47
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
56
Coomassie Stains 56
Silver Stains 57
Fluorescent Stains 57
Negative Stains 57
Stain-Free Technology 57
Detection of Proteins on Western Blots 61
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.
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.