Electrophoresis in Practice: A Guide to Methods and Applications of DNA and Protein Separations, Fourth Edition

406 Pages·2004·7.967 MB·English

by  Dr. Reiner Westermeier(auth.)

ReinerWestermeier
ElectrophoresisinPractice
ElectrophoresisinPractice,FourthEdition.ReinerWestermeier
Copyright(cid:16)2005WILEY-VCHVerlagGmbH&Co.KGaA,Weinheim
ISBN:3-527-31181-5
Related title fromWiley-VCH
ReinerWestermeier,TomNaven
Proteomicsin Practice
ALaboratoryManualofProteomeAnalysis
ISBN3-527-30354-5
Reiner Westermeier
Electrophoresis in Practice
A Guide to Methods and Applications
of DNA and Protein Separations
Fourth, revised and enlarged Edition
in collaboration with
Sonja Gronau
Phil Becket
Josef B(cid:26)lles
Hanspeter Schickle
G(cid:26)nter Theßeling
Dr.ReinerWestermeier & AllbookspublishedbyWiley-VCHarecare-
GEHealthcareBio-Sciences fullyproduced.Nevertheless,authors,
MunzingerStr.9 editors,andpublisherdonotwarrantthe
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Readersareadvisedtokeepinmindthat
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PhilBecket inaccurate.
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(cid:16)2005WILEY-VCHVerlagGmbH&Co.
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ISBN 3-527-31181-5
V
Contents
Foreword XI
Preface XIII
Abbreviations XVII
PartI Fundamentals 1
Introduction 3
1 Electrophoresis 9
1.0 General 9
1.1 Electrophoresisinnon-restrictivegels 17
1.1.1 Agarosegelelectrophoresis 17
1.1.2 Polyacrylamidegelelectrophoresisoflow-molecularweight
substances 20
1.2 Electrophoresisinrestrictivegels 21
1.2.1 TheFergusonplot 21
1.2.2 Agarosegelelectrophoresis 22
1.2.3 Polyacrylamidegelelectrophoresisofnucleicacids 24
1.2.4 Polyacrylamidegelelectrophoresisofproteins 34
2 Isotachophoresis 45
2.1 Migrationwiththesamespeed 45
2.2 “Iontrain”separation 47
2.3 Zonesharpeningeffect 47
2.4 Concentrationregulationeffect 47
3 Isoelectricfocusing 51
3.1 Principles 51
3.2 GelsforIEF 53
3.3 Temperature 54
3.4 ControllingthepHgradient 54
ElectrophoresisinPractice,FourthEdition.ReinerWestermeier
Copyright(cid:1)2005WILEY-VCHVerlagGmbH&Co.KGaA,Weinheim
ISBN:3-527-31181-5
VI Contents
3.5 ThekindsofpHgradients 55
3.5.1 Freecarrierampholytes 55
3.5.2 ImmobilizedpHgradients 59
3.6 ProteindetectioninIEFgels 62
3.7 Preparativeisoelectricfocusing 62
3.8 Titrationcurveanalysis 64
4 Blotting 67
4.1 Principle 67
4.2 Transfermethods 68
4.2.1 Diffusionblotting 68
4.2.2 Capillaryblotting 68
4.2.3 Pressureblotting 68
4.2.4 Vacuumblotting 69
4.2.5 Electrophoreticblotting 70
4.3 Blottingmembranes 72
4.4 Buffersforelectrophoretictransfers 73
4.5 Generalstaining 75
4.6 Blocking 75
4.7 Specificdetection 76
4.8 Proteinsequencing 78
4.9 Transferproblems 79
5 Interpretationofelectropherograms 81
5.1 Introduction 81
5.1.1 Puritycontrol 81
5.1.2 Quantificationprerequisites 81
5.2 Imageanalysis 83
5.2.1 Hardwareforimageanalysis 84
5.2.2 Softwareforimageanalysis 87
6 ProteomeAnalysis 91
6.1 General 91
6.2 Samplepreparation 96
6.3 Two-dimensionalelectrophoresis 100
6.4 Detectiontechniques 102
6.5 Imageanalysis 107
6.6 Proteinspotidentification 109
6.6.1 Massspectrometrymethods 111
6.6.2 Peptidemassfingerprinting 116
6.6.3 Proteincharacterization 118
6.7 Bioinformatics 118
6.8 Functionalproteomics 119
Contents VII
7 Instrumentation 121
7.1 Currentandvoltageconditions 121
7.2 Powersupply 123
7.3 Separationchambers 124
7.3.1 Verticalapparatus 124
7.3.2 Horizontalapparatus 125
7.4 Stainingapparatusforgelsandblots 128
7.5 Automatedelectrophoresis 128
7.6 Instrumentsfor2-Delectrophoresis 130
7.6.1 Isoelectricfocusingapparatus 130
7.6.2 Multipleslabgelapparatus 131
7.7 Safetymeasures 131
7.8 Environmentalaspects 132
PartII EquipmentandMethods 133
EquipmentforPartII 135
Instrumentation 136
Speciallaboratoryequipment 138
Consumables 139
Chemicals 140
Method1:PAGEofdyes 145
1 Samplepreparation 145
2 Stocksolutions 145
3 Preparingthecastingcassette 146
4 Castingultrathin-layergels 148
5 Electrophoreticseparation 149
Method2:Agaroseandimmunoelectrophoresis 151
1 Samplepreparation 151
2 Stocksolutions 152
3 Preparingthegels 152
4 Electrophoresis 157
5 Proteindetection 159
Method3:Titrationcurveanalysis 163
1 Samplepreparation 163
2 Stocksolutions 164
3 Preparingtheblankgels 164
4 Titrationcurveanalysis 167
5 Coomassieandsilverstaining 170
6 Interpretingthecurves 172
VIII Contents
Method4:NativePAGEinamphotericbuffers 175
1 Samplepreparation 176
2 Stocksolutions 177
3 Preparingtheemptygels 178
4 Electrophoresis 182
5 Coomassieandsilverstaining 185
Method5:AgaroseIEF 189
1 Samplepreparation 189
2 Preparingtheagarosegel 190
3 Isoelectricfocusing 193
4 Proteindetection 195
Method6:PAGIEFinrehydratedgels 197
1 Samplepreparation 198
2 Stocksolutions 198
3 Preparingtheblankgels 199
4 Isoelectricfocusing 201
5 Coomassieandsilverstaining 204
6 Perspectives 208
Method7:HorizontalSDS-PAGE 211
1 Samplepreparation 211
2 Stocksolutionsforgelpreparation 215
3 Preparingthecastingcassette 216
4 Gradientgel 218
5 Electrophoresis 221
6 Proteindetection 224
7 Blotting 227
8 Perspectives 227
Method8:VerticalPAGE 231
1 Samplepreparation 232
2 Stocksolutions 233
3 Singlegelcasting 233
4 Multiplegelcasting 237
5 Electrophoresis 241
6 SDSelectrophoresisofsmallpeptides 242
7 Two-dimensionalelectrophoresis 244
8 DNAelectrophoresis 244
9 Longshelflifegels 245
10 Detectionofbands 245
Contents IX
Method9:Semi-dryblottingofproteins 247
1 Transferbuffers 248
2 Technicalprocedure 249
3 Stainingofblottingmembranes 253
Method10:IEFinimmobilizedpHgradients 257
1 Samplepreparation 258
2 Stocksolutions 258
3 Immobilinerecipes 259
4 Preparingthecastingcassette 262
5 PreparingthepHgradientgels 264
6 Isoelectricfocusing 269
7 Staining 271
8 StrategiesforIPGfocusing 273
Method11:High-resolution2-Delectrophoresis 275
1 Samplepreparation 276
2 Stocksolutions 279
3 Preparingthegels 280
4 Separationconditions 285
5 Stainingprocedures 293
Method12:PAGEofdoublestrandedDNA 299
1 Stocksolutions 300
2 Preparingthegels 301
3 Samplepreparation 305
4 Electrophoresis 305
5 Silverstaining 310
Method13:NativePAGEofsinglestrandedDNA 313
1 Sampletreatment 315
2 Gelproperties 316
3 Buffersandadditives 317
4 Conditionsforelectrophoresis 318
5 StrategyforSSCPanalysis 318
Method14:Denaturinggradientgelelectrophoresis 321
1 Samplepreparation 322
2 Rehydrationsolutions 322
3 Preparingtherehydrationcassette 323
4 Rehydration 325
5 Electrophoresis 327
6 Silverstaining 329
X Contents
Method15:DenaturingPAGEofDNA 331
1 Samplepreparation 332
2 Solutions 333
3 Rehydration 333
4 Electrophoresis 334
5 Silverstaining 337
Appendix Trouble-shooting 339
A1 Isoelectricfocusing 339
A1.1 PAGIEFwithcarrierampholytes 339
A1.2 AgaroseIEFwithcarrierampholytes 348
A1.3 ImmobilizedpHgradients 352
A2 SDSelectrophoresis 359
A3 VerticalPAGE 369
A4 Semidryblotting 371
A5 2-Delectrophoresis 379
A6 DNAelectrophoresis 385
References 389
Index 399