Protein Analysis and Purification (eBook)

Preface 6
Acknowledgments 7
Contents 8
An Overview of This Manual 24
Introduction 24
Kits, Cores, and Computers 26
How to Use This Book 26
Basic Laboratory Equipment 27
Laboratory Automation 28
Beyond Protein Analysis and Purification 29
Protein Structure 31
Introduction 31
A. The Amino Acids 31
B. The Four Levels of Protein Structure 34
C. Chemical Characteristics of Proteins 40
Tracking the Target Protein 47
Introduction 47
A. Labeling Cells and Proteins 49
Metabolic Labeling Adherent Cells 51
Metabolic Labeling Cells Growing in Suspension 51
Pulse-Chase Labeling 52
Lactoperoxidase Labeling Cell Surface Proteins 54
Labeling Surface Proteins with IODO- GEN ® 55
Non-radioactive Biotinylation of Cell Surface Proteins 56
Domain-Selective Biotinylation and Streptavidin- Agarose Precipitation 57
Labeling Isolated Proteins with Chloramine T 59
B. Lysis: Preparation of the Cell Free Extract 60
Lysis of Cells in Suspension ( Continuation of Protocol 3.2) 61
Lysis of Adherent Cells ( Continuation of Protocol 3.1) 62
C. Principles of Immunoprecipitation 62
Immunoprecipitation 65
Sequential Immunoprecipitation: Dissociation and Reimmunoprecipitation of Immune Complexes 67
Eliminating Interfering Immunoglobulin Bands During IP- Western Detection: Analysis of the Immunoprecipitate under Non- Reducing Conditions 69
Nondenaturing Immunoprecipitation 70
D. Additional Methods to Identify Associated Proteins 70
Preparation of Sucrose Gradients 71
Cross-Linking Proteins Added to Cells: Analysis of Receptor- Ligand Interaction 78
Cross-Linking Proteins in Solution 78
Cross-Linking Extraneously Added Ligand to Cells 79
Cross-Linking Proteins in Solution Using the Homobifunctional Reagent Dithiobis ( succinimidyl propionate) ( DSP) 80
Electrophoretic Techniques 86
Introduction to Polyacrylamide Gel Electrophoresis ( PAGE) 86
A. Preparation of SDS-Polyacrylamide Gels 89
Assembling the Plates 89
Casting the Separating Gel 90
Casting the Stacking Gel 92
Gradient Gels 93
Sample Preparation 95
Running the Gel: Attaching the Gel Cassette to the Apparatus and Loading the Samples 96
Drying the Gel 99
Separation of Low Molecular Weight Proteins by Tricine- SDS- PAGE ( TSDS- PAGE) 100
B. 2-Dimensional (2-D) Gel Systems 102
Preparation of the Sample for Isoelectric Focusing 106
Preparation and Running of Isoelectric Focusing Tube Gels 107
Equilibration of the First-Dimension Gel or Strip 109
Measuring the pH of the Gel Slices 110
Nonequilibrium pH Gradient Electrophoresis ( NEPHGE) 112
2-D Gels—The Second Dimension: SDS- PAGE 113
Labeling Proteins with Cyanine Dyes ( Cy3 and Cy5) 114
Nonreducing-Reducing 2- D Gels 115
C. Detection of Protein Bands in Polyacrylamide Gels 118
Staining and Destaining the Gel with Coomassie Blue 119
Coomassie Staining Using GelCode® Blue 121
Staining Gels with SYPRO® Ruby 121
Silver Staining 123
Reversible Negative Staining of Proteins in Gels with Imidazole and Zinc Salts 124
Molecular Weight Determination by SDS- PAGE 126
D. Recovery of Proteins from the Gel 128
Excising the Protein Band from the Dried Gel 128
Extracting the Target Protein from the Dried Gel 129
E. Identification of Enzyme Activity in Polyacrylamide Gels 129
Localization of Proteases: Copolymerization of Substrate in the Separating Gel 130
Identification of Protease Inhibitors: Reverse Zymography 131
Locating the Enzyme Activity: Reacting the Gel with Substrate Solution after Electrophoresis 132
Detection of b–glucuronidase Activity in Polyacrylamide Gels 133
Gel Shifts 135
Getting Started with Protein Purification 141
Introduction 141
A. Making a Cell Free Extract 143
Nuclear Extracts 149
Total Lymphocyte Extract 150
Subcellular Fractionation 150
B. Protein Quantitation 151
Bradford Standard Assay 153
Bradford Microassay 155
Protein Determination Using Bicinchoninic Acid ( BCA) 156
Compatible Substances for the BCA Protein Assay 157
Incompatible Substances 157
NanoOrange® Protein Quantitation Assay: A Fluorescence- Based Assay of Proteins in Solution 158
C. Manipulating Proteins in Solution 159
Recovery of Protein by Ammonium Sulfate Precipitation 161
Preparation of Dialysis Tubing 164
D. Precipitation Techniques 166
Salting Out with Ammonium Sulfate 166
Precipitation with Acetone 168
PEG Precipitation 169
Removal of PEG from Precipitated Proteins 170
Recovery of Protein from Dilute Solutions by Methanol Chloroform Precipitation 171
Recovery of Protein by Trichloroacetic Acid ( TCA) Precipitation 172
Concentration of Proteins by Acetone Precipitation 173
Membrane Proteins 176
Introduction 176
A. Peripheral Membrane Proteins 177
Alkali Extraction 179
High pH Membrane Fractionation 180
B. Integral Membrane Proteins 181
Butanol Extraction 182
Single-Phase Butanol Extraction 182
C. Detergents 183
Differential Detergent Solubilization 192
Solubilization Trial 194
Transfer and Detection of Proteins on Membrane Supports 200
Introduction 200
A. Transfer of Proteins to Membrane Supports 200
Transfer of Proteins to Nitrocellulose or Polyvinylidene Difluoride 201
Enhanced Capture of Small Histidine Containing Polypeptides on Membranes in the Presence of ZnCl2 204
Dot Blots 205
Thin-Layer Chromatography Blotting 205
B. Staining the Blot 206
Total Protein Staining with India Ink 206
Reversible Staining with Ponceau S 207
Irreversible, Rapid Staining with Coomassie Brilliant Blue 207
Staining Immobilized Glycoproteins by Periodic Acid/ Schiff ( PAS) 208
C. Recovery of Proteins from the Blot 209
Recovery of Proteins Using an Organic Solvent System 209
Recovery of Proteins from the Blot Using a Detergent- Based Solvent System 210
Blocking the Blot 211
Exposing the Blot to Primary Antibody 211
Ligand Binding 215
Lectin Blotting 216
Bacterial Protein Overlay Analysis 218
D. Detection of the Target Protein 219
Biotinylation of Proteins 220
Purifying and Biotinylating Antibodies from Immunoblots 221
Colorimetric Detection with Diaminobenzidine, 3,3¢,4,4¢- tetraaminobiphenyl) (DAB) 222
Colorimetric Detection Using Alkaline Phosphatase 223
Enhanced Chemiluminescence 224
Stripping and Reprobing the Blot 226
Direct Autoradiography 227
Phosphorimaging 230
Identification of the Target Protein 234
Introduction 234
A. Peptide Mapping 234
Thermal Denaturation 235
B. Enzymatic Cleavage of Proteins 237
Peptide Mapping by Proteolysis and Analysis by Electrophoresis 238
Cleavage of Proteins Immobilized on the Membrane 240
Tryptic Cleavage of Protein Eluted from PVDF Membrane 241
C. Chemical Cleavage 242
Cyanogen Bromide Cleavage of Proteins on PVDF Membrane 242
N-Chlorosuccinimide (NCS) Mapping 244
Hydroxylamine Cleavage of Proteins in Polyacrylamide Gels 245
Formic Acid Cleavage 246
Chemical Cleavage at Cysteine Residues with DTNB 247
D. Microsequencing from PVDF Membranes 248
Transferring Spots from 2-D Gels to PVDF Membranes 251
Protein Hydrolysis: Total Amino Acid Composition of the Target Protein 252
Preparation of Proteins for MS Analysis 254
In-gel Tryptic Digestion 255
Extraction of Peptides from Gel Pieces Containing Integral Membrane Proteins 256
MS Basics 257
Protein Identification by MS 261
Protein Database Searches 267
The Rise of Biological Databases 268
Identifying and Analyzing Posttranslational Modifications 282
Introduction 282
A. Glycosylation 284
Chemical Deglycosylation Using Trifluoromethanesulfonic Acid ( TFMS) 288
Removal of the Oligosaccharide from the Glycoprotein with N- Glycanase 290
N-glycosidase F (GPase F) Treatment of Glycoproteins in Immunoprecipitates 291
Tunicamycin 292
Identification of O-Glycosylated Amino Acids by Alkaline b-Elimination 293
b–Elimination of O-Glycans from Glycoproteins Immobilized on Blots 294
O- Glycosidase 294
O- Glycanase 295
Endoglycosidase H 295
Desialylation with Clostridium perfringens Neuraminidase 297
Desialylation with Arthrobacter ureafaciens Neuraminidase 298
Is the Target Protein a Proteoglycan? 300
B. Phosphorylation 301
Metabolic Labeling of Cells with [32P]orthophosphate 303
Can the Target Protein Be Phosphorylated? 304
Determination of the Type of Phosphorylated Amino Acid- Immunoblotting with Anti- Phosphoamino Acid Antibodies 305
Phosphorylation of Membrane Proteins with [g 32P]GTP 306
Potato Acid Phosphatase 306
Alkaline Phosphatase 308
Immune Complex Kinase 308
Renaturation of Immobilized Kinases on PVDF Membranes 310
Phosphorylation of Substrates in SDS- Gels 312
One-Dimensional Phosphopeptide Mapping 314
Two-Dimensional Phosphopeptide Mapping 314
Isolation of Phospho-Proteins from SDS Gels: Preparation for Phosphopeptide Mapping 315
Tryptic Digestion of Isolated Phosphoproteins 316
Applying the Sample to the TLC Plate and Electrophoresis in the First Dimension 317
Second Dimension: Thin-Layer Chromatography 318
Isolation of Individual Phosphopeptides from TLC Plates 319
Phosphoamino Acid Analysis 319
Phosphoamino Acid Analysis of Phosphoproteins Isolated from PVDF Membranes 321
Identi.cation of Phosphohistidine Residues Following Heat Treatment 322
Treatment with Diethyl Pyrocarbonate 323
Treatment of Phosphorylated Membranes with HCl and NaOH 323
Sulfation 324
C. Lipid Modi.cation of Proteins 325
Identification of Palmitoylated and Myristoylated Proteins 327
Metabolic Labeling with [3H]Mevalonic Acid Derivatives 329
Enzymatic Prenylation of Recombinant Proteins 331
Preparation of Triton X-114 333
Fractionation of Integral Membrane Proteins with Triton X- 114 334
Digestion with Phosphatidylinositol Specific Phospholipase C ( PI- PLC) 335
D. Selected Modifications 337
Chromatography 347
Introduction 347
A. Important Terminology Used in Chromatography 348
B. Gel Filtration Chromatography 349
Preparation of the Gel: Hydrating and Degassing 353
Packing the Column 354
Loading Sample onto a Drained Bed 357
Spin Columns 360
Testing Fractions to Locate Protein: Bradford Spot Test 361
C. Introduction to HPLC 361
D. Ion Exchange Chromatography: Separation on the Basis of Charge 365
Selecting the Starting pH 371
Packing an Ion Exchange Column 371
Regeneration of Sephadex Ion Exchangers 374
Regeneration of Sepharose Ion Exchangers 374
Chromatofocusing 374
E. Hydrophobic Interaction Chromatography (HIC) 379
Protein Fractionation by HIC 380
Solid Phase Extraction Cartridges 381
F. Affinity Chromatography 386
Direct Antibody Coupling to Protein A Beads 388
Indirect Antibody Coupling to Protein A Beads 389
Preparation of Affinity Columns 390
Blocking the Affinity Matrix 393
Eluting the Antigen 395
Immobilization of Proteins on NHydroxysuccinimide Ester Derivatives of Agarose 396
Glycoprotein Purification Using Wheat Germ Agglutinin ( WGA) 401
Immobilized Metal Chelate Chromatography ( IMAC) 402
Purifying a Histidine Tagged Recombinant Fusion Protein 403
Hydroxylapatite Chromatography 404
Recombinant Protein Techniques 408
Introduction 408
A. In vitro Transcription and Translation 411
Preparation of the DNA Template 411
In vitro Transcription — Preparation of the mRNA 412
Guanylyltransferase Catalyzed Addition of a G(5¢)ppp(5¢)G Cap to mRNA 413
In vitro Translation: Protein Synthesis 414
Cotranslational Processing Using Canine Pancreatic Microsomal Membranes 415
Translocated Products Are Resistant to Protease Digestion 416
Was the Translational Product Glycosylated? Endoglycosidase H ( Endo H) Analysis 417
B. Recombinant Gene Products in E. coli: Expression, Identification and Characterization 419
lacZ Induction 421
Induction of the trpE Fusion Protein 421
Preparation of the Protein Extract 422
Solubilization of the Fusion Protein 423
Purification of Eukaryotic Proteins from Inclusion Bodies in E. coli 423
C. Affinity Tags 425
Production and Analysis of GST Fusion Protein Transformants ( Small Scale) 428
Purification of GST Fusion Proteins 429
Removing the GST from the Fusion Protein 430
His-Tag Purification System 431
D. Expression of Foreign Proteins in Eukaryotic Cells 438
Preparation of Protein Extracts from Yeast 439
Transfection of DNA into Eukaryotic Cells with Calcium Phosphate 444
Glycerol Shock 445
Transfection Using DEAE-Dextran 446
Stable Transfections 447
Picking Stable Colonies 448
Appendixes 454
Safety Considerations 455
First Aid: Emergency Procedures 455
Antibody Preparation 457
Production of Polyclonal Antisera in Rabbits 457
Preparation of Antigen-Adjuvant Emulsion 458
Intramuscular Immunization (IM) 458
Intradermal Immunization 459
Subcutaneous Immunization 459
Bleeding the Rabbit and Serum Preparation 459
Precipitation of IgG with Saturated Ammonium Sulfate 460
Purification of Antibody Using Protein A Affinity Columns 461
Purifying Total Ig 461
Numbering Mice 462
Solutions 463
Water 463
Molarity 464
Choosing and Preparing Buffers 464
Common Laboratory Solutions 469
Extinction Coefficients 471
Nucleic Acids 473
Spectrophotometric Conversions 473
DNA/Protein Conversions 473
Oligonucleotide Concentrations 473
Fluorometric Estimation of DNA Concentrations 473
RNA Precipitation 474
Modifications and Motifs 478
Nomenclature 478
Protein Modification Sequences 480
Protein Kinase Recognition Sequence Motifs 482
Subcellular Localization Motifs 483
Protein Databases 484
Centrifugation 487
Proteases and Proteolytic Enzyme Inhibitors 502
Preparation of Defatted BSA 504
Radioactivity 512
Manual and Machine Film Processing 513
Tissue Culture 515
Transwell Permeable Supports 515
Miscellaneous 517
Siliconizing Glassware 517
List of Suppliers, Vendors, Manufacturers 524
Index 532