Fundamental Laboratory Approaches for Biochemistry and Biotechnology

Alexander J. Ninfa is Associate Professor in the Department of Biological Chemistry at the University of Michigan and is internationally recognized for his work on the biochemical mechanisms of signal transduction and transcriptional regulation in bacteria. David P. Ballou is Professor in the Department of Biological Chemistry at the University of Michigan and is widely known for his work in rapid kinetics and the study of enzyme mechanisms involving redox coenzymes.

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 EnzymeAlkaline 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 Eq