Main description:

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.

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 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