Electrophoresis in practice: a guide to methods and applications of DNA and protein separations
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| Main Author: | |
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| Format: | Book |
| Language: | English German |
| Published: |
Weinheim
Wiley-VCH Verlag GmbH & Co. KGaA
[2016]
|
| Edition: | Fifth edition |
| Subjects: | |
| Online Access: | Inhaltsverzeichnis |
| Physical Description: | XXVI, 441 Seiten Illustrationen, Diagramme |
| ISBN: | 9783527338801 9783527695188 |
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MARC
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| 245 | 1 | 0 | |a Electrophoresis in practice |b a guide to methods and applications of DNA and protein separations |c Reiner Westermeier |
| 250 | |a Fifth edition | ||
| 264 | 1 | |a Weinheim |b Wiley-VCH Verlag GmbH & Co. KGaA |c [2016] | |
| 264 | 4 | |c © 2016 | |
| 300 | |a XXVI, 441 Seiten |b Illustrationen, Diagramme | ||
| 336 | |b txt |2 rdacontent | ||
| 337 | |b n |2 rdamedia | ||
| 338 | |b nc |2 rdacarrier | ||
| 650 | 4 | |a DNA |x Separation |v Laboratory manuals | |
| 650 | 4 | |a Electrophoresis |v Laboratory Manuals | |
| 650 | 4 | |a Electrophoresis |v Laboratory manuals | |
| 650 | 4 | |a Proteins |x Separation |v Laboratory manuals | |
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| _version_ | 1804175813573083136 |
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| adam_text | CONTENTS
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 MOLECULAR WEIGHT
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 THE WORKFLOW 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
.
73.2 OFF-GEL IFF 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 IFF 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)
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
S.3.2.2 PROTEIN ENRICHMENT BY AFFINITY BEADS 118
5.4 SAMPLES FOR HIGH-RESOLUTION 2D PAGE 118
5.4.1
CELL WASHING 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.S.2
EQUALIZER TECHNOLOGY 125
5.4.S.3
PRESEPARATION OF CELL ORGANELLES 126
5.4.S.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
6.22.3 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
6.2.52 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 SATURATION CYSTEINE 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
6.5.22 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
73.22 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
12 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
M L.L
SAMPLE PREPARATION 197
M1.2
STOCK SOLUTIONS 197
M1.3 PREPARING THE CASTING CASSETTE
198
ML.3.1
GASKET 198
ML.3.2 SLOT-FORMER
198
ML.3.3 ASSEMBLING THE GEL CASSETTE
199
M1.4 CASTING ULTRA-THIN-LAYER GELS
200
M1.5
ELECTROPHORETIC SEPARATION 201
M L.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 SAMPLE WELLS 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 IFF 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 2S5
M7.9.1 GEL CHARACTERISTICS 285
M7.9.2 SDS ELECTROPHORESIS IN WASHED 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
M 8.LL 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 GELS WITHOUT 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
M L0.4.1 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
M L0.5.1.5 REHYDRATION 332
M10.6 ISOELECTRIC FOCUSING 332
M L0.6.1 PLACING THE GEL ON THE COOLING PLATE 332
M 10.6.2 SAMPLE APPLICATION 335
M10.6.3 ELECTRODE SOLUTIONS 335
M10.6.4 FOCUSING CONDITIONS 335
M 10.6.5 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
M L0.7.4 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
M L 1.1 SAMPLE PREPARATION 342
M IL.1.1 SAMPLE CLEAN-UP 343
M L 1.2 PRELABELING OF PROTEINS WITH FLUORESCENT DYES 346
M L 1.2.1 LABELING OF ONE SAMPLE 346
M LL.2.2 DICE LABELING 347
MLL.2.2.1 EXPERIMENTAL DESIGN 347
MLL.2.2.2 SAMPLE PREPARATION 347
MLL.2.2.3 RECONSTITUTION OF THE CYDYES 348
M L 1.2.2.4 MINIMAL LABELING OF THE LYSINES 349
MLL.2.2.5 SATURATION LABELING OF THE CYSTEINES 350
MLL.2.2.6 PREPARATION FOR LOADING THE SAMPLES ONTO THE IPG STRIPS 351
MLL.2.2.7 DETECTION OF DIGE SPOTS 352
M L 1.3 STOCK SOLUTIONS FOR GEL PREPARATION 352
M11.4 PREPARING THE GELS 354
M L 1.4.1 IPG STRIPS 354
M L 1.4.2 SDS POLYACRYLAMIDE GELS 358
M11.5 SEPARATION CONDITIONS 359
M IL.5.1 FIRST DIMENSION (IPG-IEF) 359
MLL.5.1.1 IPG-IEF WITH CONVENTIONAL EQUIPMENT 360
M IL.5.1.2 IPG-IEF WITH IPG STRIP KIT (FIGURE ) 360
M IL.5.1.3 IPG-IEF IN INDIVIDUAL CERAMIC TRAYS 362
MLL.5.1.4 EQUIPMENT AND TRAYS FOR CUP LOADING 363
M L 1.5.2 EQUILIBRATION 366
M LL.5.3 SECOND DIMENSION (SDS ELECTROPHORESIS) 366
MLL.5.3.1 VERTICAL GELS 366
MLL.5.3.2 HORIZONTAL GELS 367
M11.6 STAINING PROCEDURES 370
M LL.6.1 STAINING OF MULTIPLE GELS 371
M L 1.6.2 COLLOIDAL COOMASSIE STAINING 371
MLL.6.2.1 STOCK SOLUTIONS 371
MLL.6.2.2 FIXATION SOLUTION 372
MLL.6.2.3 STAINING SOLUTION 372
M IL.6.2.4 STAINING PROCEDURE: 372
M LL.6.3 REVERSIBLE IMIDAZOLE-ZINC NEGATIVE STAINING 372
M L 1.6.4 SILVER STAINING 373
M L 1.6.4.1 MASS SPECTROMETRY ANALYSIS OF SILVER-STAINED SPOTS 374
MLL.6.4.2 BLUE TONING 374
M L 1.6.5 FLUORESCENT STAINING WITH SERVA PURPLE 374
MLL.6.5.1 STOCK SOLUTIONS 374
MLL.6.5.2 STAINING PROTOCOL 375
M IL.6.5.3 DETECTION 376
REFERENCES 377
METHOD 12 PAGE OF DNA FRAGMENTS 379
M L2.1 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
A 1.1 FREQUENT MISTAKES 393
AL.1.1 MISCALCULATION OF THE CROSS-LINKING FACTOR OF A POLYACRYLAMIDE
GEL 393
A 1.1.2 POLYMERIZATION TEMPERATURE AND TIME FOR A POLYACRYLAMIDE
GEL 393
AL.1.3 CREATING AGGREGATES IN SDS SAMPLES 394
A 1.1.4 TITRATION OF THE RUNNING BUFFER IN SDS ELECTROPHORESIS 394
AL.1.5 INCOMPLETE REMOVAL OF PBS FROM CELLS 395
A L.1.6 OVER-FOCUSING OF IPG STRIPS IN 2D PAGE
395
A L.1.6.1 PROTEIN DEGRADATION IN BASIC PH GRADIENTS
395
A L.1.6.2
THE THIOUREA EFFECT 395
A1.2 ISOELECTRIC FOCUSING
396
AL.2.1
PAGIEF WITH CARRIER AMPHOLYTES 396
AL.2.2 AGAROSE IFF WITH CARRIER AMPHOLYTES
402
AL.2.3 IMMOBILIZED PH GRADIENTS
405
A1.3 SDS ELECTROPHORESIS
410
AL.3.1 HORIZONTAL SDS-PAGE 410
AL.3.2
VERTICAL PAGE 418
A1.4
TWO-DIMENSIONAL ELECTROPHORESIS 419
A L.5 SEMI-DRY BLOTTING 426
A1.6 DNA ELECTROPHORESIS 431
INDEX 435
|
| any_adam_object | 1 |
| author | Westermeier, Reiner 1951- |
| author_GND | (DE-588)135840627 |
| author_facet | Westermeier, Reiner 1951- |
| author_role | aut |
| author_sort | Westermeier, Reiner 1951- |
| author_variant | r w rw |
| building | Verbundindex |
| bvnumber | BV043285296 |
| classification_rvk | VG 8200 WC 3440 |
| ctrlnum | (OCoLC)927844388 (DE-599)BVBBV043285296 |
| discipline | Chemie / Pharmazie Biologie |
| edition | Fifth edition |
| format | Book |
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| genre_facet | Anleitung Praktikum |
| id | DE-604.BV043285296 |
| illustrated | Illustrated |
| indexdate | 2024-07-10T07:22:15Z |
| institution | BVB |
| isbn | 9783527338801 9783527695188 |
| language | English German |
| oai_aleph_id | oai:aleph.bib-bvb.de:BVB01-028706586 |
| oclc_num | 927844388 |
| open_access_boolean | |
| owner | DE-11 DE-703 DE-20 DE-29T DE-19 DE-BY-UBM |
| owner_facet | DE-11 DE-703 DE-20 DE-29T DE-19 DE-BY-UBM |
| physical | XXVI, 441 Seiten Illustrationen, Diagramme |
| publishDate | 2016 |
| publishDateSearch | 2016 |
| publishDateSort | 2016 |
| publisher | Wiley-VCH Verlag GmbH & Co. KGaA |
| record_format | marc |
| spelling | Westermeier, Reiner 1951- Verfasser (DE-588)135840627 aut Electrophoresis in practice a guide to methods and applications of DNA and protein separations Reiner Westermeier Fifth edition Weinheim Wiley-VCH Verlag GmbH & Co. KGaA [2016] © 2016 XXVI, 441 Seiten Illustrationen, Diagramme txt rdacontent n rdamedia nc rdacarrier DNA Separation Laboratory manuals Electrophoresis Laboratory Manuals Electrophoresis Laboratory manuals Proteins Separation Laboratory manuals Elektrophorese (DE-588)4014373-9 gnd rswk-swf Biomolekül (DE-588)4135124-1 gnd rswk-swf Gelelektrophorese (DE-588)4123267-7 gnd rswk-swf 1\p (DE-588)4142527-3 Anleitung gnd-content 2\p (DE-588)4127380-1 Praktikum gnd-content Gelelektrophorese (DE-588)4123267-7 s DE-604 Biomolekül (DE-588)4135124-1 s Elektrophorese (DE-588)4014373-9 s 3\p DE-604 4\p DE-604 Erscheint auch als Online-Ausgabe, EPUB 978-3-527-69519-5 Erscheint auch als Online-Ausgabe, MOBI 978-3-527-69517-1 Erscheint auch als Online-Ausgabe, PDF 978-3-527-69516-4 DNB Datenaustausch application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=028706586&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis 1\p cgwrk 20201028 DE-101 https://d-nb.info/provenance/plan#cgwrk 2\p cgwrk 20201028 DE-101 https://d-nb.info/provenance/plan#cgwrk 3\p cgwrk 20201028 DE-101 https://d-nb.info/provenance/plan#cgwrk 4\p cgwrk 20201028 DE-101 https://d-nb.info/provenance/plan#cgwrk |
| spellingShingle | Westermeier, Reiner 1951- Electrophoresis in practice a guide to methods and applications of DNA and protein separations DNA Separation Laboratory manuals Electrophoresis Laboratory Manuals Electrophoresis Laboratory manuals Proteins Separation Laboratory manuals Elektrophorese (DE-588)4014373-9 gnd Biomolekül (DE-588)4135124-1 gnd Gelelektrophorese (DE-588)4123267-7 gnd |
| subject_GND | (DE-588)4014373-9 (DE-588)4135124-1 (DE-588)4123267-7 (DE-588)4142527-3 (DE-588)4127380-1 |
| title | Electrophoresis in practice a guide to methods and applications of DNA and protein separations |
| title_auth | Electrophoresis in practice a guide to methods and applications of DNA and protein separations |
| title_exact_search | Electrophoresis in practice a guide to methods and applications of DNA and protein separations |
| title_full | Electrophoresis in practice a guide to methods and applications of DNA and protein separations Reiner Westermeier |
| title_fullStr | Electrophoresis in practice a guide to methods and applications of DNA and protein separations Reiner Westermeier |
| title_full_unstemmed | Electrophoresis in practice a guide to methods and applications of DNA and protein separations Reiner Westermeier |
| title_short | Electrophoresis in practice |
| title_sort | electrophoresis in practice a guide to methods and applications of dna and protein separations |
| title_sub | a guide to methods and applications of DNA and protein separations |
| topic | DNA Separation Laboratory manuals Electrophoresis Laboratory Manuals Electrophoresis Laboratory manuals Proteins Separation Laboratory manuals Elektrophorese (DE-588)4014373-9 gnd Biomolekül (DE-588)4135124-1 gnd Gelelektrophorese (DE-588)4123267-7 gnd |
| topic_facet | DNA Separation Laboratory manuals Electrophoresis Laboratory Manuals Electrophoresis Laboratory manuals Proteins Separation Laboratory manuals Elektrophorese Biomolekül Gelelektrophorese Anleitung Praktikum |
| url | http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=028706586&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA |
| work_keys_str_mv | AT westermeierreiner electrophoresisinpracticeaguidetomethodsandapplicationsofdnaandproteinseparations |