Bio-Rad GS-900™ Calibrated Densitometer - Manual (Page 3)

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2-D Electrophoresis Guide Table of Contents
Chapter 6 Image Acquisition, Analysis,
and Spot Cutting
63
Finding Protein Spots of Interest
64
Image Acquisition 64
Image Analysis
65
Image Optimization, Spot Detection, and Quantitation 66
Gel Comparison 66
Data Normalization 66
Data Analysis and Reporting 67
Spot Cutting from 2-D Gels 67
Chapter 7 Identification and
Characterization of 2-D Protein Spots
69
Beyond Excision
70
Proteolytic Digestion 70
Washing 70
Reduction and Alkylation 70
In-Gel Proteolytic Digestion 70
Identification by Mass Spectrometry
71
Peptide Mass Fingerprinting 72
Tandem Mass Spectrometry (MS/MS) 73
Establishment of 2-D Databases 73
Part II: Methods
75
Chapter 8 Sample Preparation
75
Tips for Sample Preparation 76
Lysis (Cell Disruption) 76
Protein Solubilization 76
Buffers and Solutions
77
Cell Lysis and Protein Extraction Procedures 78
Suspension Cultured Human Cells 78
Monolayer Cultured Human Cells 78
Mammalian Tissue 79
Microbial Cultures 79
Plant Leaves 80
Sample Cleanup
81
Buffer Exchange (Desalting) 81
Sample Quantitation (RC DC Protein Assay)
82
Microfuge Tube Assay Protocol (1.5 ml) 82
Chapter 9 First-Dimension IEF
with IPG Strips
85
Tips for IEF
86
IPG Strip Rehydration and Sample Loading 86
Performing IEF
87
IPG Strip Rehydration in Rehydration/Equilibration Trays
Followed by IEF 87
IEF with Gel-Side Up 87
IEF with Gel-Side Down 88
Cup Loading (IEF with Gel-Side Up) 88
IPG Strip Rehydration in the Focusing Tray Followed by IEF 89
IEF Programming Recommendations
90
Chapter 10 Second-Dimension SDS-PAGE
95
Tips for SDS-PAGE
96
IPG Strip Equilibration
97
Sealing IPG Strips onto SDS-PAGE Gels 98
SDS-PAGE
99
Chapter 11 Protein Detection
101
Tips for Total Protein Staining
102
Long-Term Storage of Stained Gels 102
Total Protein Staining
103
Bio-Safe Coomassie Stain 103
Flamingo Fluorescent Gel Stain 103
Oriole Fluorescent Gel Stain 104
SYPRO Ruby Protein Gel Stain 104
Silver Stain Plus Kit 105
Chapter 12 In-Gel Trypsin Digestion
107
Tryptic Digestion Protocol
108
Reagents and Solutions 108
Destaining Gel Plugs from Silver-Stained Gels 108
(Pre-Treatment)
General Destaining Protocol 109
Reduction and Alkylation Protocol 109
Digestion Protocol 109
Extraction Protocol 109
Part III: Troubleshooting
111
Isoelectric Focusing
112
SDS-PAGE 113
Total Protein Staining
114
2-D Gel Evaluation
115
Part IV: Appendices
125
Appendix A 126
Glossary 126
Appendix B
130
References 130
Related Bio-Rad Literature 132
Appendix C 133
Ordering Information 133