Rent 9780805346138 | Biochemistry Laboratory : Modern Theory and Techniques

Summary Table of Contents

Many biochemistry lab instructors are now opting to either design their own experiments or select them from major educational journals.Biochemistry Laboratory: Modern Theory and Techniques addresses this issue by providing a flexible alternative without experimental protocols. Instead of requiring instructors to use specific experiments, the book focuses on detailed descriptions of modern techniques in experimental biochemistry and discusses the theory behind such techniques in detail. The extensive range of techniques includes chromatography, ... MORE

Introduction to the Biochemistry Laboratory

(34)

Safety in the Laboratory

(3)

Safety First

... MORE

(1)

Material Safety Data Sheets

(2)

Safe Practices in the Biochemistry Laboratory

(1)

Keeping Records and Communicating Experimental Results

(9)

The Laboratory Notebook

(2)

Details of the Experimental Write-Up

(2)

Communicating Results from Biochemistry Research

(5)

Using Biochemical Reagents and Solutions

(4)

Water Quality

(1)

Cleaning Laboratory Glassware

(1)

Solutions: Concentrations and Calculations

(2)

Preparing and Storing Solutions

(1)

Quantitative Transfer of Liquids

(5)

Pipets and Pipetting

(3)

Automatic Pipetting Devices

(2)

Statistical Analysis of Experimental Data

(12)

Defining Statistical Analysis

(1)

The Mean, Sample Deviation, and Standard Deviation

(4)

Spreadsheet Statistics

(1)

Statistical Analysis in Practice

(2)

Study Problems

(2)

Further Reading

(3)

The Computer as a Tool in Biochemistry and Molecular Biology

(20)

Computers in Biochemistry and Molecular Biology

(3)

Personal Computing in Biochemistry

(1)

The Computer and the Internet

(1)

The World Wide Web

(4)

Web Sites for Biochemistry and Molecular Biology

(4)

Applications of the Web

(13)

The Biochemical Literature

(4)

Web Directories, Tools, and Databases

(4)

Study Problems

(2)

Further Reading

(1)

Glossary for the Internet

(2)

General Laboratory Procedures

(46)

pH, Buffers, Electrodes, and Biosensors

(14)

Measurement of pH

(1)

Using the pH Electrode

(2)

Biochemical Buffers

(2)

Selection of a Biochemical Buffer

(5)

Buffer Dilutions

(1)

The Oxygen Electrode

(2)

Biosensors

(1)

Measurement of Protein Solutions

(5)

The Biuret and Lowry Assays

(2)

The Bradford Assay

(1)

The BCA Assay

(1)

The Spectrophotometric Assay

(1)

Measurement of Nucleic Acid Solutions

(2)

The Spectrophotometric Assay

(1)

Other Assays for Nucleic Acids

(2)

Techniques for Sample Preparation

(7)

Dialysis

(1)

Ultrafiltration

(4)

Lyophilization and Centrifugal Vacuum Concentration

(2)

Radioisotopes in Biochemistry

(18)

Origin and Properties of Radioactivity

(4)

Detection and Measurement of Radioactivity

(7)

Radioisotopes and Safety

(1)

Study Problems

(2)

Further Reading

(4)

Centrifugation Techniques in Biochemistry

101

(22)

Basic Principles of Centrifugation

101

(4)

Instrumentation for Centrifugation

105

(8)

Low-Speed Centrifuges

106

(1)

High-Speed Centrifuges

107

(4)

Ultracentrifuges

111

(2)

Applications of Centrifugation

113

(10)

Preparative Techniques

113

(1)

Analytical Measurements

114

(4)

Care of Centrifuges and Rotors

118

(2)

Study Problems

120

(1)

Further Reading

121

(2)

Purification and Identification of Biomolecules by Chromatography

123

(52)

Introduction to Chromatography

124

(2)

Partition versus Adsorption Chromatography

125

(1)

Paper and Thin-Layer Chromatography (Planar Chromatography)

126

(4)

Preparation of the Sorbent

126

(1)

Solvent Development

127

(1)

Detection and Measurement of Components

128

(1)

Applications of Planar Chromatography

129

(1)

Column Chromatography

130

(4)

Operation of a Chromatographic Column

131

(1)

Packing the Column

131

(1)

Loading the Column

132

(1)

Eluting the Column

132

(1)

Collecting the Eluent

133

(1)

Detection of Eluting Components

133

(1)

Ion-Exchange Chromatography

134

(6)

Ion-Exchange Resins

135

(1)

Selection of the Ion Exchanger

136

(2)

Choice of Buffer

138

(1)

Preparation of the Ion Exchanger

138

(1)

Using the Ion-Exchange Resin

138

(1)

Storage of Resins

139

(1)

Gel Exclusion Chromatography

140

(9)

Theory of Gel Filtration

140

(1)

Physical Characterization of Gel Chromatography

141

(1)

Chemical Properties of Gels

142

(1)

Selecting a Gel

142

(2)

Gel Preparation and Storage

144

(1)

Operation of a Gel Column

144

(2)

Applications of Gel Exclusion Chromatography

146

(3)

High-Performance Liquid Chromatography (HPLC)

149

(14)

Instrumentation

150

(4)

Stationary Phases in HPLC

154

(2)

Chiral Chromatography

156

(2)

The Mobile Phase

158

(1)

Solvents for HPLC Operations

158

(1)

Gradient Elution in HPLC

158

(2)

Sample Preparation and Selection of HPLC Operating Conditions

160

(1)

FPLC--A Modification of HPLC

161

(1)

Perfusion Chromatography

162

(1)

Affinity Chromatography and Immunoadsorption

163

(12)

Chromatographic Media

164

(1)

The Immobilized Ligand

165

(1)

Attachment of Ligand to Matrix

165

(3)

Immunoadsorption

168

(1)

Experimental Procedure for Affinity Chromatography

168

(2)

Study Problems

170

(1)

Further Reading

171

(4)

Characterization of Proteins and Nucleic Acids by Electrophoresis

175

(38)

Theory of Electrophoresis

176

(1)

Introduction

176

(1)

Theory and Practice

176

(1)

Methods of Electrophoresis

177

(23)

Polyacrylamide Gel Electrophoresis (PAGE)

177

(4)

Discontinuous Gel Electrophoresis

181

(3)

Sodium Dodecyl Sulfate--Polyacrylamide Gel Electrophoresis (SDS-Page)

184

(2)

Nucleic Acid Sequencing Gels

186

(1)

Agarose Gel Electrophoresis

187

(4)

Pulsed Field Gel Electrophoresis (PFGE)

191

(2)

Isoelectric Focusing of Proteins

193

(2)

Two-Dimensional Electrophoresis (2-DE) of Proteins

195

(2)

Capillary Electrophoresis (CE)

197

(2)

Immunoelectrophoresis (IE)

199

(1)

Practical Aspects of Electrophoresis

200

(13)

Instrumentation

200

(1)

Reagents

200

(1)

Staining and Detecting Electrophoresis Bands

201

(3)

Protein and Nucleic Acid Blotting

204

(1)

The Western Blot

204

(5)

Analysis of Electrophoresis Results

209

(1)

Study Problems

209

(2)

Further Reading

211

(2)

Spectroscopic Analysis of Biomolecules

213

(40)

Ultraviolet-Visible Absorption Spectrophotometry

214

(18)

Wavelength and Energy

214

(1)

Light Absorption

215

(2)

Electronic Transitions in Biomolecules

217

(2)

The Absorption Spectrum

219

(1)

The Beer-Lambert Law

220

(1)

Instrumentation

221

(4)

Applications of UV-VIS Spectroscopy

225

(7)

Fluorescence Spectrophotometry

232

(7)

Principles

232

(1)

Quantum Yield

233

(2)

Instrumentation

235

(1)

Applications of Fluorescence Spectroscopy

236

(1)

Difficulties in Fluorescence Measurements

237

(2)

Nuclear Magnetic Resonance Spectroscopy

239

(4)

NMR Theory

239

(1)

NMR in Biochemistry

239

(1)

NMR and Protein Structures

240

(3)

Mass Spectrometry

243

(3)

Ionization and Analysis of Proteins

244

(1)

MS Applications in Biochemistry

245

(1)

X-Ray Crystallography

246

(7)

Methodology of X-ray Crystallography

247

(1)

Study Problems

248

(1)

Further Reading

249

(4)

Biomolecular Interactions: Ligand Binding and Enzyme Reactions

253

(32)

Ligand-Macromolecule Interactions (Molecular Recognition)

253

(12)

Properties of Noncovalent Binding Interactions

254

(2)

Quantitative Characterization of Ligand Binding

256

(3)

Scatchard's Equation

259

(1)

Cooperative Binding of Ligands

259

(1)

Experimental Measurement of Ligand-Binding Interactions

260

(1)

The Bradford Protein Assay as an Example of Ligand Binding

261

(3)

Computer Software for Analysis of LM Binding

264

(1)

Biological Catalysis (Enzymes)

265

(20)

Classes of Enzymes

265

(2)

Kinetic Properties of Enzymes

267

(1)

Significance of Kinetic Constants

268

(2)

Inhibition of Enzyme Activity

270

(2)

Units of Enzyme Activity

272

(1)

Specific Activity

273

(1)

Design of an Enzyme Assay

273

(1)

Kinetic versus Fixed-Time Assay

274

(1)

Applications of an Enzyme Assay

275

(2)

Computer Software for Analysis of Enzyme Kinetic Data

277

(1)

Study Problems

278

(2)

Further Reading

280

(5)

Molecular Biology I: Structures and Analysis of Nucleic Acids

285

(24)

Introduction to the Nucleic Acids

285

(8)

Chemical Components of DNA and RNA

285

(3)

DNA Structure and Function

288

(3)

RNA Structure and Function

291

(2)

Laboratory Methods for Investigation of DNA and RNA

293

(16)

Isolation of Chromosomal DNA

293

(2)

Isolation of Plasmid DNA

295

(2)

Characterization of DNA

297

(2)

Ethidium Bromide Binding and Fluorescence

299

(1)

Agarose Gel Electrophoresis

300

(1)

Sequencing DNA Molecules

301

(2)

Isolation and Characterization of RNA

303

(1)

Study Problems

304

(1)

Further Reading

305

(4)

Molecular Biology II: Recombinant DNA, Molecular Cloning, and Enzymology

309

(20)

Recombinant DNA Biotechnology

309

(8)

Molecular Cloning

310

(2)

Steps for Preparing Recombinant DNA

312

(2)

Cloning Vectors

314

(3)

Important Enzymes in Molecular Biology and Biotechnology

317

(8)

The Restriction Endonucleases

317

(1)

Applications of Restriction Enzymes

318

(2)

Practical Aspects of Restriction Enzyme Use

320

(1)

The Polymerase Chain Reaction

321

(4)

Nucleic Acid Blotting

325

(4)

Study Problems

325

(1)

Further Reading

326

(3)

Protein Production, Purification, and Characterization

329

(42)

Procedures for the Purification of Proteins

329

(10)

Composition of Proteins

330

(1)

Amount of Protein versus Purity of Protein versus Expense

331

(1)

Basic Steps in Protein Purification

332

(1)

Preparation of the Crude Extract

333

(1)

Stabilization of Proteins in a Crude Extract

334

(3)

Separation of Proteins Based on Solubility Differences

337

(1)

Selective Techniques in Protein Purification

338

(1)

Production of Proteins by Expression of Foreign Genes

339

(5)

Gene Expression in Prokaryotic Organisms

340

(3)

Gene Expression in Eukaryotic Cells

343

(1)

Protein Characterization

344

(1)

Determination of Primary Structure

345

(8)

Amino Acid Composition

345

(5)

Sequencing DNA Instead of the Protein

350

(1)

Study Problems

350

(1)

Further Reading

351

(2)

Appendices

Appendix I List of Software Programs and Web Sites Useful for Each Chapter

353

(2)

Appendix II Properties of Common Acids and Bases

355

(1)

Appendix III Properties of Common Buffer Compounds

356

(1)

Appendix IV pKa and pH1 Values of Amino Acids

357

(1)

Appendix V Molecular Weight of Some Common Proteins

358

(1)

Appendix VI Common Abbreviations Used in This Text

359

(2)

Appendix VII Units of Measurement

361

(2)

Appendix VIII Table of the Elements

363

(2)

Appendix IX Answers to Selected Study Problems

365

(6)

Index

371

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Biochemistry Laboratory : Modern Theory and Techniques

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