Fundamental laboratory approaches for biochemistry and biotechnology

著者

書誌事項

Fundamental laboratory approaches for biochemistry and biotechnology

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

Wiley : c2010

2nd ed

  • : pbk

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注記

Previous ed.: 1998

Formerly CIP Uk

Includes bibliographical references and index

内容説明・目次

内容説明

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.

目次

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

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