Rodney Boyer Carving
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Rodney Boyer Carver
<ul><li><p>S e c o n d E d i t i o n</p><p>BIOCHEMISTRY LABORATORY:MODERN THEORYAND TECHNIQUES</p><p>Rodney BoyerHope College</p><p>Prentice HallBoston Columbus Indianapolis New York San Francisco Upper Saddle River</p><p>Amsterdam Cape Town Dubai London Madrid Milan Munich Paris Montral TorontoDelhi Mexico City So Paulo Sydney Hong Kong Seoul Singapore Taipei Tokyo</p></li><li><p>Editor in Chief, Chemistry: Adam JaworskiMarketing Manager: Erin GardnerProject Editor: Jennifer HartEditorial Assistant: Fran FalkMarketing Assistant: Nicola HoustonManaging Editor, Chemistry and Geosciences: Gina M. </p><p>CheselkaProject Manager, Production: Shari ToronSenior Manufacturing and Operations Manager: Nick SklitsisOperations Specialist: Maura ZaldivarArt Director: Jayne ConteCover Designer: Suzanne BehnkeSenior Technical Art Specialist: Connie Long</p><p>Art Editor: Ronda WhitsonPhoto Research Manager: Elaine SoaresPhoto Researcher: Dian LoftonText Permissions Editor: Beth WollarSenior Media Producer: Angela BernhardtMedia Producer: Lauren LaynSenior Media Production Supervisor: Liz WinerMedia Production Coordinator: Shannon KongFull-Service/Composition: PreMediaGlobalFull-Service Project Management: Greg </p><p>Johnson/PreMediaGlobalCopy Editor: Kami BevingtonCover photo credit: The Protein Data Bank archive</p><p> 2012, 2006 Pearson Education, Inc.Pearson Prentice HallPearson Education, Inc.Upper Saddle River, New Jersey 07458</p><p>All rights reserved. No part of this book may be reproduced, in any form or by any means, without permission in writing from the publisher.</p><p>Printed in the United States of America10 9 8 7 6 5 4 3 2 1</p><p>Pearson Prentice Hall is a trademark of Pearson Education, Inc.</p><p>ISBN-10: 0-13-604302-XISBN-13: 978-0-13-604302-7</p><p>Library of Congress Cataloging-in-Publication Data</p><p>Boyer, Rodney F.Biochemistry laboratory : modern theory and techniques / Rodney Boyer. 2nd ed.</p><p>p. cm.Includes index.ISBN-13: 978-0-13-604302-7ISBN-10: 0-13-604302-X</p><p>1. BiochemistryLaboratory manuals. I. Title.QD415.5.B69 2012572.078dc22</p><p>2010036761</p><p>Disclaimer: The laboratory methods described in this book have been exhaustively tested for safety and all attempts havebeen made to select the least hazardous chemicals and procedures possible. However, the author and publisher cannot beheld liable for any injury or damage that may occur during the performance of the procedures. It is assumed that before anyexperiment is initiated, a Material Safety Data Sheet (MSDS) for each chemical used will have been studied by the instructorand students to ensure its safe handling and disposal.</p><p>Front cover: The molecular structure is that of the hexameric form of the human hormone insulin, grouped around two zincions. The protein hexamer is thought to be the form in which insulin is stored in the beta cells of the pancreas and secretedinto the blood. The structure ID is 1AI0 in the Protein Data Bank.</p></li><li><p>To my wife, Christel</p></li><li><p>TABLE OF CONTENTS</p><p>Preface xiii</p><p>Acknowledgments xvii</p><p>About the Author xix</p><p>Chapter 1 INTRODUCTION TO THE BIOCHEMISTRYLABORATORY 1A. Safety in the Laboratory 2</p><p>Safety First 2</p><p>Material Safety Data Sheets 2</p><p>Safe Practices in the Biochemistry Laboratory 3</p><p>B. Keeping Records and Communicating Experimental Results 6</p><p>The Laboratory Notebook 6</p><p>Details of the Experimental Write-Up 7</p><p>Communicating Results from Biochemistry Research 9</p><p>C. Using Biochemical Reagents and Solutions 14</p><p>Water Purity 14</p><p>Cleaning Laboratory Glassware 15</p><p>Solutions: Concentrations and Calculations 15</p><p>Preparing and Storing Solutions 17</p><p>D. Quantitative Transfer of Liquids 18</p><p>Pipets and Pipetting 18</p><p>Automatic Pipetting Devices 21</p><p>E. Statistical Analysis of Experimental Data 23</p><p>Defining Statistical Analysis 23</p><p>The Mean, Sample Deviation, and Standard Deviation 24</p><p>Spreadsheet Statistics 28</p><p>Statistical Analysis in Practice 28Study Problems 30 Further Reading 32</p><p>Chapter 2 USING THE COMPUTER AND INTERNET FORRESEARCH IN BIOCHEMISTRY 35A. What Is Research and How Is It Done in Biochemistry? 35</p><p>What Is Research? 35</p><p>The Scientific Method 36</p><p>B. Using Computers in Biochemistry 38</p><p>v</p></li><li><p>Accessing the Internet 39</p><p>The World Wide Web 40</p><p>C. Web Sites Useful in Biochemistry 40</p><p>Directories, Library Resources, Databases, and Tools 40</p><p>Viewing Structures of Biomolecules 43</p><p>Searching the Biochemical Literature 44</p><p>Literature Searches on the Web 45</p><p>Sequence Homology in Proteins 47</p><p>Virtual Biochemistry Laboratories 47Study Problems 48 Further Reading 49 Computer Glossary 50</p><p>Chapter 3 GENERAL LABORATORY PROCEDURES 53A. pH, Buffers, Electrodes, and Biosensors 53</p><p>Measurement of pH 54</p><p>Using the pH Electrode 54</p><p>Biochemical Buffers 56</p><p>Selection of a Biochemical Buffer 57</p><p>Buffer Dilutions 63</p><p>The Oxygen Electrode 64</p><p>Biosensors 66</p><p>B. Measurement of Protein Solutions 67</p><p>The Biuret and Lowry Assays 67</p><p>The Bradford Assay 69</p><p>The BCA Assay 70</p><p>The Spectrophotometric Assay 70</p><p>C. Measurement of Nucleic Acid Solutions 71</p><p>The Spectrophotometric Assay 71</p><p>Other Assays for Nucleic Acids 72</p><p>D. Techniques for Sample Preparation 73</p><p>Dialysis 73</p><p>Ultrafiltration 74</p><p>Lyophilization and Centrifugal Vacuum Concentration 77</p><p>E. Radioisotopes in Biochemistry 80</p><p>Origin and Properties of Radioactivity 80</p><p>Detection and Measurement of Radioactivity 85</p><p>Radioisotopes and Safety 90Study Problems 91 Further Reading 92</p><p>vi Table of Contents</p></li><li><p>Chapter 4 CENTRIFUGATION TECHNIQUES IN BIOCHEMISTRY 95A. Basic Principles of Centrifugation 96</p><p>B. Instrumentation for Centrifugation 99</p><p>Low-Speed Centrifuges 99</p><p>High-Speed Centrifuges 101</p><p>Ultracentrifuges 105</p><p>C. Applications for Centrifugation 106</p><p>Preparative Techniques 106</p><p>Analytical Measurements 108</p><p>Care of Centrifuges and Rotors 112Study Problems 113 Further Reading 114</p><p>Chapter 5 PURIFICATION AND ANALYSIS OF BIOMOLECULESBY CHROMATOGRAPHY 115A. Introduction to Chromatography 116</p><p>Partition versus Adsorption Chromatography 117</p><p>B. Planar Chromatography (Paper and Thin-LayerChromatography) 118</p><p>Preparation of the Sorbent 118</p><p>Solvent Development 119</p><p>Detection and Measurement of Components 120</p><p>Applications of Planar Chromatography 121</p><p>Advanced Planar Chromatography 121</p><p>C. Column Chromatography 122</p><p>Operation of a Chromatographic Column 123</p><p>Packing the Column 124</p><p>Loading the Column 125</p><p>Eluting the Column 125</p><p>Collecting the Eluent 126</p><p>Detection of Eluting Components 126</p><p>D. Ion-Exchange Chromatography 126</p><p>Ion-Exchange Resins 127</p><p>Selection of the Ion Exchanger 128</p><p>Choice of Buffer 130</p><p>Preparation of the Ion Exchanger 130</p><p>Using the Ion-Exchange Resin 130</p><p>Storage of Resins 131</p><p>Table of Contents vii</p></li><li><p>E. Gel-Exclusion Chromatography 132</p><p>Theory of Gel Filtration 132</p><p>Physical Characterization of Gel Chromatography 133</p><p>Chemical Properties of Gels 133</p><p>Selecting a Gel 135</p><p>Gel Preparation and Storage 136</p><p>Operation of a Gel Column 136</p><p>Applications of Gel-Exclusion Chromatography 138</p><p>F. High-Performance Liquid Chromatography (HPLC) 140</p><p>Instrumentation 142</p><p>Stationary Phases in HPLC 144</p><p>Chiral Chromatography 148</p><p>The Mobile Phase 150</p><p>Sample Preparation and Selection of HPLC OperatingConditions 150</p><p>FPLCA Modification of HPLC 150</p><p>Perfusion Chromatography 151</p><p>G. Affinity Chromatography and Immunoadsorption 152</p><p>Chromatographic Media 153</p><p>The Immobilized Ligand 154</p><p>Attachment of Ligand to Matrix 154</p><p>Immunoadsorption 156</p><p>Experimental Procedure for Affinity Chromatography 157</p><p>H. Membrane-Based Chromatography 159Study Problems 161 Further Reading 163</p><p>Chapter 6 CHARACTERIZATION OF PROTEINS AND NUCLEICACIDS BY ELECTROPHORESIS 165A. The Theory of Electrophoresis 166</p><p>Introduction 166</p><p>Theory and Practice 166</p><p>B. Methods of Electrophoresis 167</p><p>Polyacrylamide Gel Electrophoresis (PAGE) 167</p><p>Discontinuous Gel Electrophoresis 172</p><p>Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis(SDS-PAGE) 174</p><p>Nucleic Acid Sequencing Gels 176</p><p>viii Table of Contents</p></li><li><p>Agarose Gel Electrophoresis 177</p><p>Pulsed Field Gel Electrophoresis (PFGE) 180</p><p>Isoelectric Focusing of Proteins 182</p><p>Two-Dimensional Electrophoresis (2-DE) of Proteins 184</p><p>Capillary Electrophoresis (CE) 185</p><p>Immunoelectrophoresis (IE) 186</p><p>C. Practical Aspects of Electrophoresis 188</p><p>Instrumentation 188</p><p>Reagents 189</p><p>Staining and Detecting Electrophoresis Bands 189</p><p>Protein and Nucleic Acid Blotting 192</p><p>The Western Blot 194</p><p>Analysis of Electrophoresis Results 197Study Problems 198 Further Reading 199</p><p>Chapter 7 SPECTROSCOPIC ANALYSIS OF BIOMOLECULES 201A. Ultraviolet-Visible Absorption Spectrometry 202</p><p>Wavelength and Energy 202</p><p>Light Absorption 204</p><p>Electronic Transitions in Biomolecules 205</p><p>The Absorption Spectrum 207</p><p>The Beer-Lambert Law 207</p><p>Instrumentation 209</p><p>Applications of UV-VIS Spectroscopy 212</p><p>B. Fluorescence Spectrometry 220</p><p>Principles 220</p><p>Quantum Yield 221</p><p>Instrumentation 222</p><p>Applications of Fluorescence Spectroscopy 223</p><p>Difficulties in Fluorescence Measurements 224</p><p>C. Nuclear Magnetic Resonance Spectroscopy 225</p><p>NMR Theory 226</p><p>NMR in Biochemistry 226</p><p>NMR and Protein Structures 227</p><p>D. Mass Spectrometry 230</p><p>Ionization and Analysis of Proteins 230</p><p>MS Applications in Biochemistry 232</p><p>Table of Contents ix</p></li><li><p>E. X-Ray Crystallography 233</p><p>Methodology of X-ray Crystallography 233Study Problems 234 Further Reading 235</p><p>Chapter 8 BIOMOLECULAR INTERACTIONS: LIGAND BINDINGAND ENZYME REACTIONS 239A. Ligand-Macromolecule Interactions (Molecular </p><p>Recognition) 239</p><p>Properties of Noncovalent Binding Interactions 240</p><p>Quantitative Characterization of Ligand Binding 242</p><p>Scatchards Equation 244</p><p>Cooperative Binding of Ligands 245</p><p>Experimental Measurement of Ligand-Binding Interactions 245</p><p>The Bradford Protein Assay as an Example of Ligand Binding 247</p><p>Computer Software for Analysis of LM Binding 249</p><p>B. Biological Catalysis (Enzymes) 250</p><p>Classes of Enzymes 250</p><p>Kinetic Properties of Enzymes 252</p><p>Significance of Kinetic Constants 254</p><p>Inhibition of Enzyme Activity 255</p><p>Units of Enzyme Activity 256</p><p>Specific Activity 258</p><p>Design of an Enzyme Assay 258</p><p>Kinetic versus Fixed-time Assay 259</p><p>Applications of an Enzyme Assay 260</p><p>Computer Software for Analysis of Enzyme Kinetic Data 262</p><p>Study Problems 262 Further Reading 264</p><p>Chapter 9 MOLECULAR BIOLOGY I: STRUCTURES AND ANALYSIS OF NUCLEIC ACIDS 267A. Introduction to the Nucleic Acids 268</p><p>Chemical Components of DNA and RNA 268</p><p>DNA Structure and Function 270</p><p>RNA Structure and Function 272</p><p>B. Laboratory Methods for Investigation of DNA and RNA 275</p><p>Isolation of Chromosomal DNA 275</p><p>Isolation of Plasmid DNA 277</p><p>x Table of Contents</p></li><li><p>Characterization of DNA 279</p><p>Ethidium Bromide Binding and Fluorescence 280</p><p>Agarose Gel Electrophoresis 282</p><p>Sequencing DNA Molecules 282</p><p>Isolation and Characterization of RNA 284Study Problems 285 Further Reading 286</p><p>Chapter 10 MOLECULAR BIOLOGY II: RECOMBINANT DNA,MOLECULAR CLONING, AND ENZYMOLOGY 289A. Recombinant DNA Biotechnology 290</p><p>Molecular Cloning 290</p><p>Steps for Preparing Recombinant DNA 292</p><p>Cloning Vectors 294</p><p>B. Important Enzymes in Molecular Biology and Biotechnology 297</p><p>The Restriction Endonucleases 297</p><p>Applications of Restriction Enzymes 298</p><p>Practical Aspects of Restriction Enzyme Use 299</p><p>The Polymerase Chain Reaction 301</p><p>C. Nucleic Acid Blotting 304Study Problems 304 Further Reading 305</p><p>Chapter 11 PROTEIN PRODUCTION, PURIFICATION, AND CHARACTERIZATION 307A. Procedures for the Purification of Proteins 308</p><p>Composition of Proteins 308</p><p>Amount of Protein versus the Purity of Protein versusExpense 308</p><p>Basic Steps in Protein Purification 309</p><p>Preparation of the Crude Extract 311</p><p>Stabilization of Proteins in a Crude Extract 312</p><p>Separation of Proteins Based on Solubility Differences 315</p><p>Selective Techniques in Protein Purification 316</p><p>B. Production of Proteins by Expression of Foreign Genes 317</p><p>Gene Expression in Prokaryotic Organisms 317</p><p>Gene Expression in Eukaryotic Cells 320</p><p>C. Protein Characterization 322</p><p>D. Determination of Primary Structure 323</p><p>Table of Contents xi</p></li><li><p>Amino Acid Composition 323</p><p>Sequencing DNA Instead of the Protein 328Study Problems 328 Further Reading 328</p><p>Appendix I List of Software Programs and Web Sites Useful for Each Chapter 331</p><p>Appendix II Properties of Common Acids and Bases 334</p><p>Appendix III Properties of Common Buffer Compounds 335</p><p>Appendix IV pKa Values and pHI Values of Amino Acids 337</p><p>Appendix V Molecular Weight of Some Common Proteins 338</p><p>Appendix VI Common Abbreviations Used in This Text 339</p><p>Appendix VII Units of Measurement 342</p><p>Appendix VIII Table of the Elements 344</p><p>Appendix IX Answers to Odd-Numbered Study Problems 348</p><p>Index 353</p><p>xii Table of Contents</p></li><li><p>PREFACE</p><p>TO THE STUDENT AND INSTRUCTOR</p><p>A biochemistry laboratory course, now offered at most colleges and universitiesin the world, is an essential component in the training of students for careers inbiochemistry, molecular biology, chemistry, and related molecular life sciencessuch as cell biology, neurosciences, and genetics. Both the American Society forBiochemistry and Molecular Biology (ASBMB) and the American ChemicalSociety (ACS) highly recommend that biochemistry majors complete such acourse. Biochemistry lab courses provide students the knowledge and skillsneeded for future research participation at the undergraduate and graduatelevel, and for jobs in the biotechnological and pharmaceutical industry.</p><p>The purpose of this book is to serve as a resource to enhance student learn-ing of theories, techniques, and methodologies practiced in the biochemistryteaching and research lab. The extensive availability of laboratory experimentspublished in journals and the desire of instructors to design their own projectsand teaching styles have lessened the need for laboratory manuals. Lab instruc-tors are especially eager to introduce new student-centered education methodssuch as problem-based learning (PBL), research-based learning, ProcessOriented Guided Inquiry Learning (POGIL), and other active-learning stylesinto their labs. However, because published experiments and homemade labmanuals usually contain only procedures, there is an increased need for a com-panion text like this one to explain the theories and principles that underpinlaboratory activities.</p><p>WHATS NEW IN THIS EDITION?</p><p>Student learning will be enhanced by the following additions and changes:</p><p> New, cutting-edge topics introduced include membrane-based chromatog-raphy (Chapter 5), less toxic electrophoresis dyes (Chapter 6), nanodropspectrophotometric analysis (Chapter 7), and using gene synthesis in pro-tein expression (Chapter 11).</p><p> An entirely rewritten section on using computers and the Internet inbiochemistry (Chapter 2).</p><p> New content on how to conduct research in biochemistry and relatedmolecular life sciences (Chapter 2).</p><p> An increase in the number of end-of-chapter study problems and a neworganization of answers in Appendix IX.</p><p> Chapter openers that begin with a content listing of topics and pagenumbers, which make it easier for students to find specific topics.</p><p> An increase in the number of study exercises within chapters so studentscan readily check their knowledge on a topic before they move on to a newtopic.</p><p>xiii</p></li><li><p> Completely updated references including books, journal articles, and espe-cially Web sites at the end of each chapter.</p><p> Updates to all three appendices located on the books Compan..</p></li></ul>
Rodney Boyer
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