Description
Book SynopsisNinfa/Ballou/Benore is a solid biochemistry lab manual, dedicated to developing research skills,allowing students to learn techniques and develop the the critical thinking and organizational approaches necessary to conduct laboratory research.
Ninfa/Ballou/Benore focuses on basic biochemistry laboratory techniques butalsoincludes molecular biology exercises, a reflection of most courses which concentrate on traditional biochemistry experiments and techniques. The experiments are designed so that theory and technique are learned as fundamental research tools, and the biochemistry and molecular biology applications are seamlessly integrated throughout the manual. The manual also includes an introduction to ethics in the laboratory, uncommon in similar manuals. Most importantly, perhaps, is the authors' three-pronged approach to encouraging students to think like a research scientist: first, the authors introduce the scientific method and the hypothesis as a framework for developing c
Table of Contents
Preface xv
Chapter 1: Getting Started in Scientific Research 1
1.1 The Difference Between Experiments and Demonstrations 1
1.2 Philosophy and Design of Experiments 3
1.3 Designing Informative Experiments 4
1.4 Ethics in Science 5
1.5 Keeping a Laboratory Notebook 9
1.6 Laboratory Reports 13
1.7 Presentation and Analysis of Data 15
1.8 The Minisymposium 18
Chapter 2: Basic Procedures in the Biochemistry Laboratory 21
2.1 Laboratory Safety 21
2.2 Special Safety Procedures are Required for Using Radioactive Materials or Operating the Autoclave 26
2.3 Measurement of Weights, Volumes, and pH 30
2.4 Various Instruments Used 38
2.5 Other General Techniques 45
2.6 Solutions and Dilutions 50
2.7 Buffers and pH 52
2.8 Appendix Calculating Titration Curves for Polyprotic Acids and Other Multiple Binding Site Receptors 62
2.9 Equipment Used in This Course 64
Chapter 3: Spectroscopic Methods 65
3.1 Introduction 65
3.2 Design and Properties of Spectrophotometers 69
3.3 Effects of Spectral Bandpass and Stray Light 72
3.4 Recording Spectrophotometers 74
3.5 Fluorescence Spectroscopy 75
3.6 Chromogenic and Fluorogenic Reactions Used for Analysis 83
3.7 Other Spectroscopic Techniques 84
3.8 Mass Spectrometry (MS) 94
Experiments 3-1 to 3-4 99
Reagents Needed for Chapter 3 103
Chapter 4: Quantification of Protein Concentration 105
4.1 Purposes of Protein Quantification 105
4.2 Factors to Consider in Choosing an Assay 107
4.3 Non-Colorimetric Procedures for Quantification of Proteins 108
4.4 Colorimetric Procedures for Quantification of Proteins 110
Experiment 4-1 115
Reagents Needed for Chapter 4 119
Chapter 5: Chromatography 121
5.1 Introduction 121
5.2 Gel-Filtration (Size Exclusion or Gel-Permeation) Chromatography 125
5.3 Affinity Chromatography 133
5.4 Ion-Exchange Chromatography 143
5.5 Hydrophobic Interaction Chromatography 149
Experiments 5-1 and 5-2 152
Reagents Needed for Chapter 5 160
Chapter 6: Gel Electrophoresis of Proteins 161
6.1 Process of Electrophoresis 161
6.2 Polyacrylamide Gels 163
6.3 SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE) of Proteins 165
6.4 Detection of Proteins in SDS-Polyacrylamide Gels 171
6.5 Applications of SDS-PAGE 173
Experiments 6-1 and 6-2 183
Reagents Needed for Chapter 6 190
Chapter 7: Overview of Protein Purification 191
7.1 Introduction 191
7.2 Development of a Suitable Assay Procedure 193
7.3 Time, Temperature, and Yield 195
7.4 Selection of the Best Source Material 195
7.5 Solubilization of the Protein 198
7.6 Initial Steps of Purification 199
7.7 Developing a Series of High-Resolution Chromatographic Steps 201
7.8 Methods Used to Change Buffer and Concentrate Protein Samples 204
7.9 A Logical Series of Steps 205
7.10 Storage of the Purified Protein 205
7.11 The Protein Purification Table 206
Chapter 8: Subcellular Fractionation 209
8.1 Introduction 209
8.2 Structural Organization of Prokaryotic and Eukaryotic Cells 210
8.3 Overview of Fractionation Protocols 213
Experiment 8-1 220
Reagents Needed for Chapter 8 227
Chapter 9: Isolation and Characterization of an Enzyme—Alkaline Phosphatase From Escherichia Coli 229
9.1 Objectives 229
9.2 Introduction and Basic Principles 229
9.3 Purification of Alkaline Phosphatase 233
9.4 Characterization of Purified Alkaline Phosphatase 242
Appendix 9-1 Assay of Alkaline Phosphatase 243
Reagents and Equipment Needed for Chapter 9 247
Chapter 10: Enzyme Kinetics 251
10.1 Why Use Steady-State Kinetics? 251
10.2 Steady-State Kinetics Principles 252
10.3 The Significance of Km and Vmax 255
10.4 Graphical Analysis 257
10.5 Competitive, “Noncompetitive, ” and “Uncompetitive” Inhibitors 260
Experiments 10-1 to 10-3 267
Reagents Needed for Chapter 10 273
Chapter 11: Ligand Binding 275
11.1 Ligand Binding is the Key to Most Biological Processes 275
11.2 Analysis of Ligand Binding at Equilibrium 277
11.3 Digression on Regression 283
11.4 Effects of the Concentrations of L and R 283
11.5 Effects of Two Sites and Cooperative Behavior 285
11.6 Analysis of the Kinetics of Ligand Binding 292
11.7 Methods Used to Study Receptor-Ligand Interactions 295
Experiments 11-1 and 11-2 302
Reagents Needed for Chapter 11 308
Chapter 12: Enzymatic Methods of Analysis 309
12.1 Enzymatic Analysis of Substrates 309
12.2 Assays for Enzymatic Activity 310
12.3 Practical Considerations 311
12.4 Coupled Assays 312
12.5 Experiments with Pyridine Nucleotide-Requiring Enzymes 314
Experiments 12-1 to 12-4 317
Reagents Needed for Chapter 12 328
Appendix 12-1: Sample Calculations 330
Chapter 13: Recombinant DNA Techniques 337
13.1 Introduction 337
13.2 Properties of Nucleic Acids 339
13.3 Strategy of Recombinant DNA Techniques 341
13.4 Cutting and Splicing DNA 343
13.5 Gel Electrophoresis of DNA 347
13.6 Introducing DNA into Cells 353
13.7 Identifying Transformed Cells 354
13.8 Vectors, Hosts, and Libraries for Recombinant DNA Experiments 359
13.9 Applications of Recombinant DNA Technology 366
Experiments 13-1 to 13-4 375
Reagents Needed for Chapter 13 386
Chapter 14: Polymerase Chain Reaction (PCR) Technology 389
14.1 Introduction 389
14.2 Principle of the PCR Method 390
14.3 The Three Steps of PCR are Controlled by Temperature 392
14.4 Applications of PCR 394
14.5 Legal and Ethical Issues 410
Experiments 14-1 to 14-6 412
Reagents Needed for Chapter 14 423
Chapter 15: Using the Computer and the Internet for Biochemical Research and Communication 425
15.1 Introduction to the World Wide Web (the Internet) 425
15.2 Information on the Internet Useful to Biochemists 431
15.3 Literature and Data Searches 432
15.4 Visualization of Molecular Structures 440
15.5 Other Useful Information 442
15.6 Homework Assignment 445
Index 447
