Basic Methods for the Biochemical Lab (eBook)

Preface 6
Table of Contents 7
Abbreviations 14
1 Quantitative Methods 16
1.1 Quantitative Determinations of Proteins 16
1.2 Quantitative Determination of Nucleic Acids 28
1.3 Quantitative Phosphate Determinations 32
1.4 Monosaccharide Determination 34
1.5 Calculations in Quantitative Analysis 35
2 Electrophoresis 38
2.1 Polyacrylamide Gel Electrophoresis Systems 38
2.2 Agarose and Paper Electrophoresis 60
2.3 Aid in Electrophoresis 64
2.4 Staining Protocols 68
2.5 Electroelution from Gels 81
2.6 Drying of Electrophoresis Gels 94
2.7 Autoradiography of Radioactive Labeled Compounds in Gels 95
3 Chromatography 98
3.1 Thin-Layer Chromatography 98
3.2 Hints for Column Chromatography of Proteins 104
3.3 Gel Permeation Chromatography ( GPC Gel Filtration, GF
3.4 Ion Exchange Chromatography (IEC) 117
3.5 Hydrophobic Interaction Chromatography (HIC) 122
3.6 Affinity Chromatography (AC) 124
3.7 Concentration of Diluted Protein Solutions 139
4 Immunochemical Protocols 144
4.1 Conjugation of Haptens (Peptides) to Carrier Proteins 144
4.2 Immunization of Laboratory Animals 158
4.3 Ammonium Sulfate Fractionation of Immunoglobulins 159
4.4 Removal of Unspecific Immunoreactivities 161
4.5 Preparation of Egg Yolk IgY Fraction 163
4.6 Antibody Fragmentation 164
4.7 HEIDELBERGER Curve (Precipitin Curve) 165
4.8 OUCHTERLONY Double-Radial Immunodiffusion 166
4.9 Immunoprecipitation of Antigens 168
4.10 Immunoelectrophoresis 169
4.11 Counterelectrophoresis 170
4.12 Dot-Blot Assay 171
4.13 Enzyme Immunosorbent Assay (EIA, ELISA) 172
5 Centrifugation 176
5.1 Speed vs Centrifugal Force Graphs 176
5.2 Differential Centrifugation 179
5.3 Density Gradient Centrifugation 180
6 Radioactive Labeling 196
6.1 Radioactive Decay 197
6.2 Decay Tables for 32-Phosphorus, 35-Sulfur, and 125- Iodine 198
6.3 Enzymatic [32P]-Phosphate Incorporation into Proteins 200
6.4 Iodination with [125I]-Iodine Reagents 202
6.5 Scintillation Cocktails for Liquid Scintillation Counting 203
7 Buffers 206
7.1 Theoretical Considerations 206
7.2 Plot for Buffer Calculations 213
7.3 pH Indicators 214
7.4 Buffer Recipes 214
8 Tables 224
8.1 Concentration Units 224
8.2 Conversion Factors for SI Units 225
8.3 Data of Frequently Used Substances 227
8.4 Protein Data 231
8.5 Protease Inhibitors 236
8.6 Single-Letter Codes and Molecular Masses of Amino Acids 237
8.7 Spectroscopic Data of Nucleotides 240
8.8 Detergents (“Surfactants”) 240
8.9 Refractive Index and Density of Sucrose Solutions 243
8.10 Ammonium Sulfate Saturation Table 244
8.11 Diluted Solutions 246
8.12 Mixture Rule 247
9 Statistics and Data Analysis 248
9.1 Statistical Equations 248
9.2 Data Analysis 252
9.3 Diagnostic Sensitivity and Specificity 259
9.4 Software for the Lab 259
Subject Index 262