Electrophoresis in Practice A Guide to Methods and Applications of DNA and Protein Separations
, by Westermeier, Reiner
- ISBN: 9783527338801 | 3527338802
- Cover: Hardcover
- Copyright: 5/16/2016
This fifth edition of the successful, long-selling classic has been completely revised and expanded, omitting some topics on obsolete DNA electrophoresis, but now with a completely new section on electrophoretic micro-methods and on-the-chip electrophoresis.
The text is geared towards advanced students and professionals and contains extended background sections, protocols and a trouble-shooting section. It is now also backed by a supplementary website providing all the figures for teaching purposes, as well as a selection of animated figures tested in many workshops to explain the underlying principles of the different electrophoretic methods.
The text is geared towards advanced students and professionals and contains extended background sections, protocols and a trouble-shooting section. It is now also backed by a supplementary website providing all the figures for teaching purposes, as well as a selection of animated figures tested in many workshops to explain the underlying principles of the different electrophoretic methods.
Reiner Westermeier worked after PhD graduation and Post-doc at the Technische Universitat Munchen for 30 years as a specialist for electrophoresis methods for leading bioanalytics and biotechnology companies. His area of responsibility included co-operation in product development, writing of scientic papers and method instructions, trouble shooting in customer laboratories, performing seminars and practical courses on electrophoresis and proteomics, as well as giving scientific talks at congresses on a world-wide basis. He is editor and author of several books, e.g. 'Electrophoresis in Practice' (in German and in English), 'Proteomics in Practice', and 'Difference Gel Electrophoresis'.
Foreword XIX
Abbreviations, Symbols, Units XXI
Preface XXV
Part I Fundamentals 1
Introduction 1
Principle 1
Areas of Applications 3
The Sample 3
The Buffer 4
Electroendosmosis 5
References 6
1 Electrophoresis 7
1.1 General 7
1.1.1 Electrophoresis in Free Solution 7
1.1.2 Electrophoresis in Supporting Media 12
1.1.3 Gel Electrophoresis 13
1.1.3.1 Gel Types 13
1.1.3.2 Instrumentation for Gel Electrophoresis 17
1.1.3.3 Current and Voltage Conditions 17
1.1.4 Power Supply 19
1.1.5 Separation Chambers 20
1.1.5.1 Vertical Systems 20
1.1.5.2 Horizontal Systems 21
1.2 Electrophoresis in Nonrestrictive Gels 25
1.2.1 Agarose Gel Electrophoresis 25
1.2.1.1 Zone Electrophoresis 25
1.2.1.2 Immunoelectrophoresis 26
1.2.1.3 Affinity Electrophoresis 27
1.2.2 Polyacrylamide Gel Electrophoresis of Low MolecularWeight Substances 28
1.3 Electrophoresis in Restrictive Gels 28
1.3.1 The Ferguson Plot 28
1.3.2 Agarose Gel Electrophoresis 29
1.3.2.1 Proteins 29
1.3.2.2 Nucleic Acids 29
1.3.3 Pulsed-Field Gel Electrophoresis 30
1.3.4 Polyacrylamide Gel Electrophoresis of Nucleic Acids 32
1.3.4.1 DNA Sequencing 32
1.3.4.2 DNA Typing 34
1.3.4.3 Mutation Detection Methods 35
1.3.4.4 Denaturing PAGE of Microsatellites 37
1.3.4.5 Two-dimensional DNA Electrophoresis 37
1.3.5 Polyacrylamide Gel Electrophoresis of Proteins 37
1.3.5.1 Disc Electrophoresis 37
1.3.5.2 Gradient Gel Electrophoresis 39
1.3.5.3 SDS Electrophoresis 40
1.3.5.4 Cationic Detergent Electrophoresis 47
1.3.5.5 Blue Native Electrophoresis 47
1.3.5.6 Rehydrated Polyacrylamide Gels 48
1.3.5.7 Two-Dimensional Electrophoresis Techniques 49
1.3.5.8 GeLC-MS 50
References 51
2 Isotachophoresis 57
2.1 Migration with the Same Speed 57
2.2 "Ion Train" Separation 59
2.3 Zone Sharpening Effect 59
2.4 Concentration Regulation Effect 59
2.5 Quantitative Analysis 60
References 61
3 Isoelectric Focusing 63
3.1 Principles 63
3.2 Gels for IEF 65
3.2.1 Polyacrylamide Gels 65
3.2.2 Agarose Gels 67
3.3 Temperature 68
3.4 Controlling the pH Gradient 68
3.5 Kinds of pH Gradients 69
3.5.1 Free Carrier Ampholytes 69
3.5.1.1 Electrode Solutions 70
3.5.1.2 Denaturing IEF: Urea IEF 71
3.5.1.3 Separator IEF 72
3.5.1.4 Plateau Phenomenon 73
3.5.1.5 TheWorkflow of a Carrier Ampholyte IEF Run 73
3.5.2 Immobilized pH Gradients (IPG) 73
3.5.2.1 Preparation of Immobilized pH Gradients 75
3.5.2.2 Applications of Immobilized pH Gradients 76
3.6 Protein Detection in IEF Gels 77
3.7 Preparative Isoelectric Focusing 77
3.7.1 Carrier Ampholyte IEF in Gel 77
3.7.2 Carrier Ampholyte IEF in Free Solution 78
3.7.3 Immobilized pH Gradients 78
3.7.3.1 Isoelectric Membranes 78
3.7.3.2 Off-Gel IEF 79
3.8 Titration Curve Analysis 80
References 82
4 High-Resolution Two-Dimensional Electrophoresis 85
4.1 IEF in Immobilized pH Gradient Strips 85
4.1.1 Strip Lengths 86
4.1.2 pH Gradient Types 86
4.1.3 The Influence of Salts and Buffer Ions on the Separation 87
4.1.4 Basic IPG Gradients 88
4.1.5 Advantages of Immobilized pH Gradient Strips in 2D Electrophoresis 89
4.1.6 Rehydration of IPG Strips 90
4.1.6.1 Basic pH Gradients 90
4.1.6.2 Reswelling Tray 91
4.1.6.3 Cover Fluid 91
4.1.6.4 Rehydration Time 92
4.1.7 Sample Application on IPG Strips 92
4.1.8 IEF Conditions 95
4.1.8.1 Electrode Pads 95
4.1.8.2 Temperature 95
4.1.8.3 Electric Conditions 95
4.1.8.4 Time 96
4.1.9 Instrumentation 96
4.1.9.1 The Strip Tray Accessory 97
4.1.9.2 Dedicated Instruments for IPG Strips 97
4.1.9.3 Running IEF in IPG Strips 97
4.2 SDS-PAGE 98
4.2.1 Equilibration of the IPG Strips 98
4.2.2 Technical Concepts for the Second Dimension (SDS-PAGE) 99
4.2.2.1 Vertical Set-ups 99
4.2.2.2 Horizontal Set-ups 99
4.2.3 Gel Types 101
4.2.3.1 Gel Sizes 101
4.2.3.2 Vertical Gels 101
4.2.3.3 Horizontal Gels 102
4.2.4 Gel Casting 102
4.2.4.1 Gels for Multiple Vertical Systems 102
4.2.4.2 Gels for Horizontal Systems 104
4.2.5 Running the SDS Gels 105
4.2.5.1 Vertical Systems 105
4.2.5.2 Horizontal Systems 106
4.3 Proteomics 106
References 108
5 Protein Sample Preparation 111
5.1 Protein Quantification Methods 111
5.2 Preparation of Native Samples 112
5.3 Samples for SDS Electrophoresis 113
5.3.1 SDS Treatment 113
5.3.1.1 Nonreducing SDS Treatment 114
5.3.1.2 Reducing SDS Treatment 115
5.3.1.3 Reducing SDS Treatment with Subsequent Alkylation 116
5.3.2 Clean-up and Protein Enrichment 117
5.3.2.1 Precipitation 117
5.3.2.2 Protein Enrichment by Affinity Beads 118
5.4 Samples for High-Resolution 2D PAGE 118
5.4.1 CellWashing 119
5.4.2 Cell Disruption 119
5.4.3 Sample Acquisition and Storage 119
5.4.4 Protease Inactivation 122
5.4.5 Phosphatase Inactivation 122
5.4.6 Alkaline Conditions 123
5.4.7 Removal of Contaminants 123
5.4.7.1 Precipitation Methods 123
5.4.7.2 Affinity Beads 125
5.4.8 Prefractionation 125
5.4.8.1 Depletion of Highly Abundant Proteins 125
5.4.8.2 Equalizer Technology 125
5.4.8.3 Preseparation of Cell Organelles 126
5.4.8.4 Prefractionation according to Isoelectric Points 126
5.4.9 Special Case: Plant Proteins 127
References 127
6 Protein Detection 131
6.1 Fixation 131
6.1.1 IEF Gels 132
6.1.2 Agarose Gels 132
6.1.3 SDS Polyacrylamide Gels 132
6.2 Poststaining Methods 133
6.2.1 Organic Dyes 133
6.2.1.1 Monodisperse Coomassie Brilliant Blue Staining 133
6.2.1.2 Colloidal Coomassie Brilliant Blue Staining 133
6.2.1.3 Acid Violet 17 Staining for IEF Gels 134
6.2.2 Silver Staining 134
6.2.2.1 Colloidal Silver Staining 134
6.2.2.2 Silver Nitrate Staining 134
6.2.2.3 Ammoniacal Silver Staining 135
6.2.3 Negative Staining 136
6.2.3.1 Copper Staining 136
6.2.3.2 Imidazole Zinc Staining 136
6.2.4 Fluorescent Staining 136
6.2.5 Specific Detection 138
6.2.5.1 Proteins with Posttranslational Modifications 138
6.2.5.2 Isoenzymes 139
6.2.6 Stain-Free Technology 140
6.3 Prelabeling 140
6.3.1 Prelabeling with Fluorescent Tags 140
6.3.2 Radioactive Labeling of Living Cells 141
6.3.3 Labeling with Stable Isotopes 141
6.4 Difference Gel Electrophoresis (DIGE) 143
6.4.1 Minimum Lysine Labeling 143
6.4.2 SaturationCysteine Labeling 144
6.4.3 The Internal Standard 146
6.4.4 Experimental Design 147
6.4.5 Major Benefits of 2D DIGE 147
6.4.6 Specific Labeling of Cell-Surface Proteins 148
6.4.7 Comparative Fluorescence Gel Electrophoresis 148
6.5 Imaging, Image Analysis, Spot Picking 149
6.5.1 Quantitative Evaluation 149
6.5.1.1 Quantification Prerequisites 149
6.5.1.2 Critical Issues in Quantification 150
6.5.2 Imaging Systems 151
6.5.2.1 Optical Density 152
6.5.2.2 Densitometry 152
6.5.2.3 CCD Cameras 153
6.5.3 Image Analysis 154
6.5.3.1 One-Dimensional Gel Software 155
6.5.3.2 Two-Dimensional Gel Software 156
6.5.4 Protein Identification and Characterization 158
6.5.4.1 Spot-Picking 159
References 160
7 Blotting 165
7.1 Transfer Methods 165
7.1.1 Diffusion Blotting 165
7.1.2 Capillary Blotting 165
7.1.3 Pressure Blotting 166
7.1.4 Vacuum Blotting 167
7.1.5 Electrophoretic Blotting 168
7.1.5.1 Tank Blotting 168
7.1.5.2 Semidry Blotting 169
7.1.5.3 Electrophoretic Blotting of Film-Backed Gels 171
7.2 Blotting Membranes 171
7.3 Buffers for Electrophoretic Transfers 172
7.3.1 Proteins 172
7.3.1.1 Tank Blotting 172
7.3.1.2 Semidry Blotting 173
7.3.2 Nucleic Acids 174
7.3.2.1 Tank Blotting 174
7.3.2.2 Semidry Blotting 174
7.4 General Staining 174
7.5 Blocking 175
7.6 Specific Detection 175
7.6.1 Hybridization 175
7.6.2 Enzyme Blotting 176
7.6.3 Immunoblotting 176
7.6.4 Lectin Blotting 179
7.6.5 Stripping, Reprobing 179
7.6.6 Double Blotting 180
7.7 Protein Sequencing 180
7.8 Transfer Issues 180
7.9 Electro-Elution of Proteins from Gels 181
References 183
Part II Equipment and Methods 187
Equipment 187
Methods 187
Small Molecules 187
Proteins 187
DNA 188
Instrumentation 188
Accessories 189
Consumables 190
8 Special Laboratory Equipment 191
9 Consumables 193
10 Chemicals 195
10.1 Reagents 195
Method 1 PAGE of Dyes 197
M1.1 Sample Preparation 197
M1.2 Stock Solutions 197
M1.3 Preparing the Casting Cassette 198
M1.3.1 Gasket 198
M1.3.2 Slot-Former 198
M1.3.3 Assembling the Gel Cassette 199
M1.4 Casting Ultra-Thin-Layer Gels 200
M1.5 Electrophoretic Separation 201
M1.5.1 Removing the Gel from the Cassette 201
Method 2 Agarose and Immunoelectrophoresis 205
M2.1 Sample Preparation 205
M2.2 Stock Solutions 206
M2.3 Preparing the Gels 206
M2.3.1 Agarose Gel Electrophoresis 206
M2.3.1.1 Preparing the Slot-Former 207
M2.3.1.2 Assembling the Gel Cassette 207
M2.3.2 Immunoelectrophoresis Gels 209
M2.3.2.1 Punching Out the SampleWells and Troughs 210
M2.4 Electrophoresis 211
M2.4.1 Grabar–Williams Technique 212
M2.4.2 Laurell Technique 212
M2.5 Protein Detection 214
M2.5.1 Coomassie Staining (Agarose Electrophoresis) 214
M2.5.2 Immunofixing of Agarose Electrophoresis 214
M2.5.3 Coomassie Staining (Immunoelectrophoresis) 215
M2.5.4 Silver Staining 215
References 216
Method 3 Titration Curve Analysis 217
M3.1 Sample Preparation 217
M3.2 Stock Solutions 217
M3.3 Preparing the Blank Gels 218
M3.3.1 Preparing the Casting Cassette 218
M3.3.2 Assembling the Gel Cassette 219
M3.3.3 Filling the Gel Cassette 220
M3.3.4 Removing the Gel from the Cassette 221
M3.3.5 Washing the Gel 221
M3.4 Titration Curve Analysis 222
M3.4.1 Reswelling the Rehydratable Gel 222
M3.4.2 Formation of the pH Gradient 222
M3.4.3 Native Electrophoresis in the pH Spectrum 223
M3.5 Coomassie and Silver Staining 224
M3.5.1 Colloidal Coomassie Staining 224
M3.5.2 Acid Violet 17 Staining 224
M3.5.3 Five-Minute Silver Staining of Dried Gels 225
M3.6 Interpreting the Curves 225
References 227
Method 4 Native PAGE in Amphoteric-Buffers 229
M4.1 Sample Preparation 230
M4.2 Stock Solutions 230
M4.3 Preparing the Empty Gels 231
M4.3.1 Slot-Former 231
M4.3.2 Assembling the Casting Cassette 232
M4.3.3 Polymerization Solutions 233
M4.3.4 Filling the Cooled Gel Cassette 234
M4.3.5 Removing the Gel from the Casting Cassette 234
M4.3.6 Washing the Gel 234
M4.4 Electrophoresis 235
M4.4.1 Rehydration in Amphoteric Buffers 235
M4.5 Coomassie and Silver Staining 240
M4.5.1 Colloidal Coomassie Staining 240
M4.5.2 Acid Violet 17 Staining 240
M4.5.3 Five-Minute Silver Staining of Dried Gels 241
References 242
Method 5 Agarose IEF 243
M5.1 Sample Preparation 243
M5.2 Preparing the Agarose Gel 244
M5.2.1 Making the Spacer Plate Hydrophobic 244
M5.2.2 Assembling the Casting Cassette 244
M5.2.3 Preparation of Electrode Solutions 246
M5.3 Isoelectric Focusing 247
M5.4 Protein Detection 249
M5.4.1 Coomassie Blue Staining 249
M5.4.2 Immunofixation 249
M5.4.3 Silver Staining 250
References 251
Method 6 PAGIEF in Rehydrated Gels 253
M6.1 Sample Preparation 253
M6.2 Stock Solutions 254
M6.3 Preparing the Blank Gels 254
M6.3.1 Making the Spacer Plate Hydrophobic 254
M6.3.2 Assembling the Casting Cassette 255
M6.3.3 Filling the Gel Cassette 256
M6.3.4 Removing the Gel from the Casting Cassette 257
M6.3.5 Washing the Gel 257
M6.4 Isoelectric Focusing 257
M6.4.1 Rehydration Solution with Carrier Ampholytes (SERVALYT™, Pharmalyte™) 257
M6.4.2 Reswelling the Gel 257
M6.4.3 Separation of Proteins 259
M6.4.4 Sample Application 259
M6.5 Coomassie and Silver Staining 260
M6.5.1 Colloidal Coomassie Staining 260
M6.5.2 Acid Violet 17 Staining 261
M6.5.3 Five-Minute Silver Staining of Dried Gels 261
M6.5.4 The Most Sensitive Silver Staining Procedure for IEF 262
M6.6 Perspectives 264
References 266
Method 7 Horizontal SDS-PAGE 267
M7.1 Sample Preparation 267
M7.1.1 Nonreducing SDS Treatment 267
M7.1.2 Reducing SDS Treatment 268
M7.1.3 Reducing SDS Treatment with Alkylation 268
M7.2 Prelabeling with Fluorescent Dye 269
M7.2.1 Labeling 269
M7.2.2 Detection 269
M7.3 Stock Solutions for Gel Preparation 270
M7.4 Preparing the Casting Cassette 271
M7.4.1 Preparing the Slot-Former 271
M7.4.2 Assembling the Casting Cassette 272
M7.5 Gradient Gel 273
M7.5.1 Pouring the Gradient 273
M7.6 Electrophoresis 277
M7.6.1 Preparing the Separation Chamber 277
M7.6.2 Placing the Gel on the Cooling Plate 277
M7.6.3 Electrophoresis 278
M7.7 Protein Detection 279
M7.7.1 Hot Coomassie Staining 279
M7.7.2 Colloidal Staining 280
M7.7.2.1 Stock Solutions 280
M7.7.2.2 Fixation Solution 280
M7.7.2.3 Staining Solution 280
M7.7.2.4 Staining Procedure 281
M7.7.3 Reversible Imidazole–Zinc Negative Staining 281
M7.7.4 Silver Staining 281
M7.7.4.1 Blue Toning 282
M7.7.5 Fluorescent Staining with SERVA Purple 283
M7.7.5.1 Stock Solutions 283
M7.7.5.2 Staining Protocol 283
M7.7.5.3 Detection 284
M7.8 Blotting 284
M7.9 Perspectives 285
M7.9.1 Gel Characteristics 285
M7.9.2 SDS Electrophoresis inWashed and Rehydrated Gels 285
M7.9.3 SDS Disc Electrophoresis in a Rehydrated and Selectively Equilibrated Gel 285
M7.9.4 Peptide Separation 286
References 287
Method 8 Vertical PAGE 289
M8.1 Sample Preparation and Prelabeling 290
M8.2 Stock Solutions for SDS- PAGE 290
M8.3 Single Gel Casting 291
M8.3.1 Discontinuous SDS-Polyacrylamide Gels 292
M8.3.2 Porosity Gradient Gels 293
M8.4 Multiple Gel Casting 295
M8.4.1 Multiple Discontinuous SDS Polyacrylamide Gels 296
M8.4.2 Multiple SDS Polyacrylamide Gradient Gels 298
M8.5 Electrophoresis 299
M8.5.1 Running Conditions 300
M8.6 SDS Electrophoresis of Small Peptides 301
M8.7 Blue Native PAGE 303
M8.8 Two-Dimensional Electrophoresis 306
M8.9 DNA Electrophoresis 307
M8.10 Long-Shelf-Life Gels 308
M8.11 Protein Detection 308
M8.12 Preparing Glass Plates with Bind-Silane 308
M8.12.1 Coating a Glass Plate with Bind-Silane 309
M8.12.2 Removal of Gel and Bind-Silane from a Glass Plate 309
References 310
Method 9 Semidry Blotting of Proteins 311
M9.1 Transfer Buffers 313
M9.2 Technical Procedure 314
M9.2.1 GelsWithout Support Film 315
M9.2.2 Gels on Film Backing 315
M9.2.2.1 Using a Nitrocellulose (NC) Blotting Membrane 316
M9.2.2.2 Using a PVDF Blotting Membrane 316
M9.2.2.3 Transfer from Cut-Off Gels 317
M9.3 Staining of Blotting Membranes 318
References 320
Method 10 IEF in Immobilized pH Gradients 321
M10.1 Sample Preparation 322
M10.2 Stock Solutions 322
M10.3 Immobiline Recipes 323
M10.3.1 Custom-Made pH Gradients 323
M10.4 Preparing the Casting Cassette 327
M10.4.1 Making the Spacer Plate Hydrophobic 327
M10.4.2 Assembling the Casting Cassette 327
M10.5 Preparing the pH Gradient Gels 328
M10.5.1 Pouring the Gradient 328
M10.5.1.1 Setting Up the Casting Apparatus 328
M10.5.1.2 Filling the Cassette 329
M10.5.1.3 Washing the Gel 331
M10.5.1.4 Storage 332
M10.5.1.5 Rehydration 332
M10.6 Isoelectric Focusing 332
M10.6.1 Placing the Gel on the Cooling Plate 332
M10.6.2 Sample Application 335
M10.6.3 Electrode Solutions 335
M10.6.4 Focusing Conditions 335
M10.6.5 Measuring the pH Gradient 336
M10.7 Staining 336
M10.7.1 Colloidal Coomassie Staining 336
M10.7.2 Acid Violet 17 Staining 337
M10.7.3 Staining Procedure 337
M10.7.4 Silver Staining 337
M10.7.5 Practical Tip 337
M10.8 Strategies for IPG Focusing 337
References 339
Method 11 High-Resolution 2D Electrophoresis 341
M11.1 Sample Preparation 342
M11.1.1 Sample Clean-Up 343
M11.2 Prelabeling of Proteins with Fluorescent Dyes 346
M11.2.1 Labeling of One Sample 346
M11.2.2 DIGE Labeling 347
M11.2.2.1 Experimental Design 347
M11.2.2.2 Sample Preparation 347
M11.2.2.3 Reconstitution of the CyDyes 348
M11.2.2.4 Minimal Labeling of the Lysines 349
M11.2.2.5 Saturation Labeling of the Cysteines 350
M11.2.2.6 Preparation for Loading the Samples onto the IPG Strips 351
M11.2.2.7 Detection of DIGE Spots 352
M11.3 Stock Solutions for Gel Preparation 352
M11.4 Preparing the Gels 354
M11.4.1 IPG Strips 354
M11.4.2 SDS Polyacrylamide Gels 358
M11.5 Separation Conditions 359
M11.5.1 First Dimension (IPG-IEF) 359
M11.5.1.1 IPG-IEF with Conventional Equipment 360
M11.5.1.2 IPG-IEF with IPG Strip Kit (Figure ) 360
M11.5.1.3 IPG-IEF in Individual Ceramic Trays 362
M11.5.1.4 Equipment and Trays for Cup Loading 363
M11.5.2 Equilibration 366
M11.5.3 Second Dimension (SDS Electrophoresis) 366
M11.5.3.1 Vertical Gels 366
M11.5.3.2 Horizontal Gels 367
M11.6 Staining Procedures 370
M11.6.1 Staining of Multiple Gels 371
M11.6.2 Colloidal Coomassie Staining 371
M11.6.2.1 Stock Solutions 371
M11.6.2.2 Fixation Solution 372
M11.6.2.3 Staining Solution 372
M11.6.2.4 Staining Procedure: 372
M11.6.3 Reversible Imidazole–Zinc Negative Staining 372
M11.6.4 Silver Staining 373
M11.6.4.1 Mass Spectrometry Analysis of Silver-Stained Spots 374
M11.6.4.2 Blue Toning 374
M11.6.5 Fluorescent Staining with SERVA Purple 374
M11.6.5.1 Stock Solutions 374
M11.6.5.2 Staining Protocol 375
M11.6.5.3 Detection 376
References 377
Method 12 PAGE of DNA Fragments 379
M12.1 Stock Solutions 380
M12.2 Preparing the Gels 381
M12.3 Sample Preparation 385
M12.4 Electrophoresis 386
M12.5 Silver Staining 391
Appendix Troubleshooting 393
A1.1 Frequent Mistakes 393
A1.1.1 Miscalculation of the Cross-Linking Factor of a Polyacrylamide Gel 393
A1.1.2 Polymerization Temperature and Time for a Polyacrylamide Gel 393
A1.1.3 Creating Aggregates in SDS Samples 394
A1.1.4 Titration of the Running Buffer in SDS Electrophoresis 394
A1.1.5 Incomplete Removal of PBS from Cells 395
A1.1.6 Over-focusing of IPG Strips in 2D PAGE 395
A1.1.6.1 Protein Degradation in Basic pH Gradients 395
A1.1.6.2 The "Thiourea Effect" 395
A1.2 Isoelectric Focusing 396
A1.2.1 PAGIEF with Carrier Ampholytes 396
A1.2.2 Agarose IEF with Carrier Ampholytes 402
A1.2.3 Immobilized pH Gradients 405
A1.3 SDS Electrophoresis 410
A1.3.1 Horizontal SDS-PAGE 410
A1.3.2 Vertical PAGE 418
A1.4 Two-Dimensional Electrophoresis 419
A1.5 Semi-Dry Blotting 426
A1.6 DNA Electrophoresis 431
Index 435
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