by
Angelika Görg（被英国电泳学会授予“双向电泳皇后”称号的德国女科学家）
Andreas Klaus, Carsten Lück, Florian Weiland
and Walter Weiss
Technische Universität München

Table of Contents
1 INTRODUCTION ............................................................................................................................. 1
1.1 Two-dimensional electrophoresis with IPGs (IPG-Dalt) ........................................................ 2
1.2 The IPG-Dalt protocol: Principle ............................................................................................... 2
2 SAMPLE PREPARATION .............................................................................................................. 6
2.1 Sample preparation and protein solubilization ....................................................................... 6
2.2 Sample prefractionation procedures ..................................................................................... 11
PROTOCOLS ............................................................................................................................. 14
I. Extraction and solubilization of cell and tissue samples .......................................................... 14
II. Sample prefractionation using Sephadex IEF ........................................................................ 19
3 2-D ELECTROPHORESIS WITH IMMOBILIZED pH GRADIENTS (IPG-DALT) ........................ 23
3.1 First dimension: IEF with immobilized pH gradients (IPGs) ............................................... 24
3.1.1 IPG gel casting. Procedure and recipes ........................................................................ 24
PROTOCOL ............................................................................................................................... 25
IPG gel casting ............................................................................................................................ 25
3.1.2 IPG strip rehydration and sample application .............................................................. 29
PROTOCOLS ............................................................................................................................. 31
I. Rehydration of IPG DryStrips with sample solution ................................................................. 32
II. Rehydration of IPG DryStrips for sample cup-loading ............................................................ 33
3.1.3 IEF using IPG strips from pH 2.5 to 12. General aspects and guidelines .................. 35
PROTOCOLS ............................................................................................................................. 40
I. IEF on the Multiphor apparatus. Running conditions for different IPGs .................................. 40
II. IEF using the IPGphor unit. Running conditions for different IPGs ........................................ 45
3.2 IPG strip equilibration for proper protein transfer and pattern quality .............................. 50
PROTOCOL ............................................................................................................................... 51
IPG strip equilibration .................................................................................................................. 51
3.3 Second dimension: SDS-PAGE on horizontal or vertical systems ..................................... 53
3.3.1 SDS gel casting ................................................................................................................ 54
PROTOCOLS ............................................................................................................................. 54
I. Casting of horizontal SDS gels on plastic backing .................................................................. 56
II. Casting of vertical SDS gels for multiple runs ......................................................................... 57
3.3.2 Running SDS gels ............................................................................................................ 60
-v -
PROTOCOLS ............................................................................................................................. 61
I. SDS electrophoresis of horizontal gels on plastic backing ...................................................... 61
Ia. SDS-PAGE on laboratory-made gels .................................................................................. 61
Ib. SDS-PAGE on ready-made Excel gels ................................................................................ 63
II. Multiple vertical SDS electrophoresis ..................................................................................... 64
IIa. SDS-PAGE using the Ettan Dalt II vertical electrophoresis unit ......................................... 64
IIb. SDS-PAGE using the Hoefer Dalt electrophoresis unit ...................................................... 65
4 PROTEIN DETECTION AND QUANTITATION ........................................................................... 67
4.1 Universal protein detection and quantitation methods ....................................................... 68
4.2 Analysis of post-translational modifications (PTMs) ........................................................... 71
4.3 Difference gel electrophoresis (DIGE) for differential display and quantitation ............... 74
PROTOCOLS ............................................................................................................................. 76
I. Coomassie Brilliant Blue (CBB) staining .................................................................................. 77
II. Silver staining .......................................................................................................................... 78
III: SYPRO Ruby fluorescent stain .............................................................................................. 80
IV: Pro-Q Diamond phosphoprotein stain ................................................................................... 81
V: Pro-Q Emerald glycoprotein stain ........................................................................................... 82
VI: Fluorescent Difference Gel Electrophoresis (DIGE) ............................................................. 83
VII: Blotting ................................................................................................................................. 87
5 COMPUTERIZED 2-D IMAGE ANALYSIS AND DATABASE CONSTRUCTION ...................... 91
5.1 Computer-assisted 2-D image analysis ................................................................................. 92
5.2 Two-dimensional electrophoresis databases ....................................................................... 92
6 PROTEIN IDENTIFICATION FROM 2-D GEL SPOTS ................................................................ 93
PROTOCOL ............................................................................................................................... 94
Protein Identification by MALDI-ToF-MS .................................................................................... 95
7 PITFALLS .................................................................................................................................... 96
8 SUMMARIZED PROTOCOL OF IPG-DALT ................................................................................ 97
APPENDIX I: Trouble shooting ..................................................................................................... 98
APPENDIX II: Sample preparation for 2D gel electrophoresis ................................................ 107
APPENDIX III: Detection of proteins on 2-D gels ...................................................................... 122
APPENDIX IV: Image analysis of 2D protein patterns ............................................................. 137
REFERENCES ............................................................................................................................... 149