The Protein Protocols Handbook

Cover......Page 1
Title......Page 4
Preface......Page 6
Contents......Page 7
Contributors......Page 15
1. Protein Determination by UV Absorption......Page 19
2. The Lowry Method for Protein Quantitation......Page 24
3. The Bicinchoninic Acid (BCA) Assay for Protein Quantitation......Page 27
4. The Bradford Method for Protein Quantitation......Page 31
5. Ultrafast Protein Determinations Using Microwave Enhancement......Page 37
6. Quantitation of Tryptophan in Proteins......Page 45
7. Protein Quantitation Using Flow Cytometry......Page 48
8. Copper Iodide Staining of Proteins on Solid Phases......Page 54
9. Kinetic Silver Staining of Proteins Adsorbed to Microtiter Plates......Page 59
10. Nondenaturing Polyacrylamide Gel Electrophoresis of Proteins......Page 62
11. SDS Polyacrylamide Gel Electrophoresis of Proteins......Page 67
12. Gradient SDS Polyacrylamide Gel Electrophoresis of Proteins......Page 74
13. Cetyltrimethylammonium Bromide Discontinuous Gel Electrophoresis of Proteins......Page 78
14. Acetic Acid-Urea Polyacrylamide Gel Electrophoresis of Basic Proteins......Page 93
15. Acid-Urea-Triton Polyacrylamide Gels for Histones......Page 101
16. SDS-Polyacrylamide Gel Electrophoresis of Peptides......Page 111
17. Isoelectric Focusing of Proteins in Ultra-Thin Polyacrylamide Gels......Page 118
18. Isoelectric Focusing Under Denaturing Conditions......Page 123
19. Radioisotopic Labeling of Proteins for Polyacrylamide Gel Electrophoresis......Page 128
20. Two-Dimensional PAGE Using Carrier Ampholyte pH Gradients in the First Dimension......Page 134
21. Two-Dimensional PAGE Using Immobilized pH Gradients......Page 140
22. Two-Dimensional PAGE Using Flat-Bed IEF in the First Dimension......Page 145
23. Free Zone Capillary Electrophoresis......Page 149
24. Capillary Isoelectric Focusing with Electro-Osmotic Flow Mobilization......Page 161
25. Quantification of Radiolabeled Proteins in Polyacrylamide Gels......Page 167
26. Quantification of Proteins by Staining in Polyacrylamide Gels......Page 172
27. Quantitation of Proteins Separated by Electrophoresis Using Coomassie Brilliant Blue......Page 178
28. Rapid Staining of Proteins in Polyacrylamide Gels with Nile Red......Page 184
29. Zn2+-Reverse Staining of Proteins in Polyacrylamide Gels......Page 191
30. Protein Staining with Calconcarboxylic Acid in Polyacrylamide Gels......Page 200
31. Detection of Proteins and Sialoglycoproteins in Polyacrylamide Gels Using Eosin Y Stain......Page 205
32. Electroelution of Proteins from Polyacrylamide Gels......Page 208
33. High-Performance Electrophoresis Chromatography......Page 216
34. Drying Polyacrylamide Gels......Page 224
35. Detection of Proteins in Polyacrylamide Gels by Silver Staining......Page 229
36. Autoradiography and Fluorography of Acrylamide Gels......Page 234
37. Protein Blotting by Electroblotting......Page 242
38. Protein Blotting by the Semidry Method......Page 246
39. Protein Blotting by the Capillary Method......Page 258
40. Protein Blotting of Basic Proteins Resolved on Acid-Urea-Triton-Polyacrylamide Gels......Page 260
41. Alkaline Phosphatase Labeling of Antibody Using Glutaraldehyde......Page 265
42. β-Galactosidase Labeling of Antibody Using MBS......Page 267
43. Horseradish Peroxidase Labeling of Antibody Using Periodate Oxidation......Page 269
44. Protein Staining and Immunodetection Using Colloidal Gold......Page 271
45. Fluorescent Protein Staining on Nitrocellulose with Subsequent Immunodetection of Antigen......Page 284
46. Coupling of Antibodies with Biotin......Page 288
47. Preparation of Avidin Conjugates......Page 297
48. Detection of Polypeptides on Blots Using Secondary Antibodies or Protein A......Page 307
49. Detection of Proteins on Blots Using Avidin- or Streptavidin-Biotin......Page 316
50. Detection of Proteins on Blots Using Chemiluminescent Systems......Page 321
51. Carboxymethylation of Cysteine Using IodoacetamideIodoacetic Acid......Page 328
52. Performic Acid Oxidation......Page 331
53. Succinylation of Proteins......Page 333
54. Pyridylethylation of Cysteine Residues......Page 335
55. Side-Chain Selective Chemical Modifications of Proteins......Page 338
56. Nitration of Tyrosines......Page 341
57. Ethoxyformylation of Histidine......Page 344
58. Modification of Arginine Side Chains with p -Hydroxyphenylglyoxal......Page 346
59. Amidation of Carboxyl Groups......Page 348
60. Amidination of Lysine Side Chains......Page 350
61. Modification of Tryptophan with 2-Hydroxy-5-Nitrobenzylbromide......Page 352
62. Modification of Sulfhydryl Groups with DTNB......Page 354
63. Chemical Cleavage of Proteins at Methionyl Residues......Page 356
64. Chemical Cleavage of Proteins at Tryptophan Residues......Page 361
65. Chemical Cleavage of Proteins at Aspartyl Residues......Page 367
66. Chemical Cleavage of Proteins at Cysteinyl Residues......Page 371
67. Chemical Cleavage of Proteins at Asparaginyl-Glycyl Peptide Bonds......Page 374
68. Preparation of Peptides for Microsequencing from Proteins in Polyacrylamide Gels......Page 378
69. In Situ Chemical and Enzymatic Digestions of Proteins Immobilized on Miniature Hydrophobic Columns......Page 384
70. Enzymatic Digestion of Membrane-Bound Proteins for Peptide Mapping and Internal Sequence Analysis......Page 389
71. Enzymatic Digestion of Proteins in Solution and in SDS Polyacrylamide Gels......Page 399
72. Reverse-Phase HPLC Separation of Enzymatic Digests of Proteins......Page 410
73. Peptide Mapping by Two-Dimensional Thin-Layer Electrophoresis-Thin-Layer Chromatography......Page 418
74. Peptide Mapping by Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis......Page 429
75. Peptide Mapping by High-Performance Liquid Chromatography......Page 434
76. Production of Protein Hydrolysates Using Enzymes......Page 437
77. Amino Acid Analysis Using Precolumn Derivatization with 6-Aminoquinolyl- n -Hydroxysuccinimidyl Carbamate......Page 441
78. Amino Acid Analysis Using Precolumn Derivatization with Phenylisothiocyanate......Page 446
79. Molecular-Weight Estimation for Native Proteins Using Size-Exclusion High-Performance Liquid Chromatography......Page 452
80. Detection of Disulfide-Linked Peptides by HPLC......Page 458
81. Diagonal Electrophoresis for Detecting Disulfide Bridges......Page 460
82. Estimation of Disulfide Bonds Using Ellman’s Reagent......Page 465
83. Quantitation of Cysteine Residues and Disulfide Bonds by Electrophoresis......Page 467
84. Detection of Disulfide-Linked Peptides by Mass Spectrometry......Page 473
85. Analyzing Protein Phosphorylation......Page 478
86. Identification of Proteins Modified by Protein (D -AspartylL -Isoaspartyl) Carboxyl Methyltransferase......Page 484
87. Analysis of Protein Palmitoylation......Page 493
88. Removal of Pyroglutamic Acid Residues from the N-Terminus of Peptides and Proteins......Page 500
89. The Dansyl Method for Identifying N-Terminal Amino Acids......Page 503
90. The Dansyl-Edman Method for Peptide Sequencing......Page 509
91. Matrix-Assisted Laser Desorption Ionization Mass Spectrometry as a Complement to Internal Protein Sequencing......Page 514
92. A Manual C-Terminal Sequencing Procedure for Peptides......Page 529
93. C-Terminal Sequence Analysis with Carboxypeptidase Y......Page 541
94. Rapid Epitope Mapping by Carboxypeptidase Digestion and Immunoblotting......Page 544
95. Epitope Mapping of a Protein Using the Geysen (PEPSCAN) Procedure......Page 551
96. Epitope Mapping of Protein Antigens by Competition ELISA......Page 564
97. Identification of Glycoproteins on Nitrocellulose Membranes Using Lectin Blotting......Page 570
98.  A Lectin-Binding Assay for the Rapid Characterization of the Glycosylation of Purified Glycoproteins......Page 586
99. Staining of Glycoproteins Proteoglycans on SDS-Gels......Page 593
100. Chemical Methods of Analysis of Glycoproteins......Page 598
101. Monosaccharide Analysis by HPAEC......Page 600
102. Monosaccharide Analysis by GC......Page 603
103. Determination of Monosaccharide Linkage and Substitution Patterns by GC-MS Methyiation Analysis......Page 605
104. Sialic Acid Analysis by HPAEC-PAD......Page 608
105. Chemical Release of O -Linked Oligosaccharide Chains......Page 610
106. O -Linked Oligosaccharide Profiling by HPLC......Page 612
107. O -Linked Oligosaccharide Profiling by HPAEC-PAD......Page 614
108. Release of N -Linked Oligosaccharide Chains by Hydrazinolysis......Page 616
109. Enzymatic Release of O - and N -Linked Oligosaccharide Chains......Page 620
110. N -Linked Oligosaccharide Profiling by HPLC on Porous Graphitized Carbon (PGC)......Page 622
111. N -Linked Oligosaccharide Profiling by HPAEC-PAD......Page 624
112. The Chloramine T Method for Radiolabeling Protein......Page 626
113. The Lactoperoxidase Method for Radiolabeling Protein......Page 630
114. The Bolton and Hunter Method for Radiolabeling Protein......Page 632
115. The Iodogen Method for Radiolabeling Protein......Page 634
116. Purification and Assessment of Quality of Radioiodinated Protein......Page 636
117. Conjugation of Peptides to Carrier Proteins via Glutaraldehyde......Page 640
118. Conjugation of Peptide to Carrier Proteins via m -Maleimidobenzoyl- N -Hydroxysuccinimide Ester (MBS)......Page 649
119. Conjugation of Peptides to Carrier Protein via Carbodiimide......Page 653
120. Raising of Polyclonal Antisera......Page 655
121. Elution of SDS-PAGE Separated Proteins from Immobilon Membranes for Use as Antigens......Page 659
122. Production of Highly Specific Polyclonal Antibodies Using a Combination of 2D Electrophoresis and Nitrocellulose-Bound Antigen......Page 663
123. Production and Characterization of Antibodies Against Peptides......Page 671
124. Production of Antibodies Using Proteins in Gel Bands......Page 677
125. Purification of IgG by Precipitation with Sodium Sulfate or Ammonium Sulfate......Page 681
126. Purification of IgG Using Caprylic Acid......Page 683
127. Purification of IgG Using DEAE-Sepharose Chromatography......Page 684
128. Purification of IgG Using Ion-Exchange HPLC FPLC......Page 686
129. Purification of IgG by Precipitation with Polyethylene Glycol (PEG)......Page 689
130. Purification of IgG Using Protein A or Protein G......Page 690
131. Purification of IgG Using Gel-Filtration Chromatography......Page 692
132. Purification of IgG Using Affinity Chromatography on Antigen-Ligand Columns......Page 695
133. Purification of IgG Using Thiophilic Chromatography......Page 698
134. Analysis of IgG Fractions by Electrophoresis......Page 700
135. Ouchterlony Double Immunodiffusion......Page 703
136. Single Radial Immunodiffusion......Page 707
137. Rocket Immunoelectrophoresis......Page 710
138. Two-Dimensional Immunoelectrophoresis......Page 716
139. Immunogen Preparation and Immunization Procedures for Rats and Mice......Page 722
140. Hybridoma Production......Page 725
141. Screening Hybridoma Culture Supernatants Using Solid-Phase Radiobinding Assay......Page 728
142. Screening Hybridoma Culture Supernatants Using ELISA......Page 730
143. Growth and Purification of Murine Monoclonal Antibodies......Page 732
144. A Rapid Method for Generating Large Numbers of High-Affinity Monoclonal Antibodies from a Single Mouse......Page 734
Index......Page 744