Fundamental Laboratory Approaches for Biochemistry and Biotechnology / Edition 2

by Alexander J. Ninfa, David P. Ballou, Marilee Benore

Overview

Ninfa/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 but also includes 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 conclusive experiments; second, the manual’s experiments are designed to become increasingly complex in order to teach more advanced techniques and analysis; finally, gradually, the students are required to devise their own protocols.   In this way, students and instructors are able to break away from a “cookbook” approach and to think and investigate for themselves.

Suitable for lower-level and upper-level courses; Ninfa spans these courses and can also be used for some first-year graduate work.

Product Details

ISBN-13:	9780470087664
Publisher:	Wiley
Publication date:	05/26/2009
Edition description:	2nd ed.
Pages:	480
Product dimensions:	8.40(w) x 10.80(h) x 0.70(d)

About the Author

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.

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 K_m and V_max 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