Biochemistry Laboratory : Modern Theory and Techniques

by Boyer, Rodney F.

• ISBN13: 9780805346138
• ISBN10: 0805346139
• Edition: 1st
• Format: Paperback
• Copyright: 2006-01-01
• Publisher: Prentice Hall

Summary

KEY BENEFIT: 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 Techniquesaddresses 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. Part I: Theory and Experimental Techniques,Introduction to the Biochemistry Laboratory, The Computer as a Tool in Biochemistry and Molecular Biology, General Laboratory Procedures, Centrifugation Techniques in Biochemistry, Purification and Identification of Biomolecules by Chromatography, Characterization of Proteins and Nucleic Acids by Electrophoresis, Spectroscopic Analysis of Biomolecules, Biomolecular Interactions: Ligand Binding and Enzyme Reactions, Molecular Biology I: Structures and Analysis of Nucleic Acids, Molecular Biology II: Recombinant DNA. Molecular Cloning, and Enzymology, Protein Production, Purification, and Characterization, Part II: Teaching the Biochemistry/Molecular Biology Lab, A Brief History, A Variety of Teaching Methods, Essential BMB Concepts and Skills for Student Learning, Experiments in Biochemistry and Molecular BiologyKEY MARKET: For all readers interested in laboratory experiments.

Table of Contents

Introduction to the Biochemistry Laboratory	1	(34)
Safety in the Laboratory	2	(3)
Safety First	2	(1)
Material Safety Data Sheets	2	(2)
Safe Practices in the Biochemistry Laboratory	4	(1)
Keeping Records and Communicating Experimental Results	5	(9)
The Laboratory Notebook	5	(2)
Details of the Experimental Write-Up	7	(2)
Communicating Results from Biochemistry Research	9	(5)
Using Biochemical Reagents and Solutions	14	(4)
Water Quality	14	(1)
Cleaning Laboratory Glassware	15	(1)
Solutions: Concentrations and Calculations	15	(2)
Preparing and Storing Solutions	17	(1)
Quantitative Transfer of Liquids	18	(5)
Pipets and Pipetting	18	(3)
Automatic Pipetting Devices	21	(2)
Statistical Analysis of Experimental Data	23	(12)
Defining Statistical Analysis	23	(1)
The Mean, Sample Deviation, and Standard Deviation	24	(4)
Spreadsheet Statistics	28	(1)
Statistical Analysis in Practice	28	(2)
Study Problems	30	(2)
Further Reading	32	(3)
The Computer as a Tool in Biochemistry and Molecular Biology	35	(20)
Computers in Biochemistry and Molecular Biology	35	(3)
Personal Computing in Biochemistry	36	(1)
The Computer and the Internet	37	(1)
The World Wide Web	38	(4)
Web Sites for Biochemistry and Molecular Biology	38	(4)
Applications of the Web	42	(13)
The Biochemical Literature	42	(4)
Web Directories, Tools, and Databases	46	(4)
Study Problems	50	(2)
Further Reading	52	(1)
Glossary for the Internet	53	(2)
General Laboratory Procedures	55	(46)
pH, Buffers, Electrodes, and Biosensors	55	(14)
Measurement of pH	56	(1)
Using the pH Electrode	56	(2)
Biochemical Buffers	58	(2)
Selection of a Biochemical Buffer	60	(5)
Buffer Dilutions	65	(1)
The Oxygen Electrode	66	(2)
Biosensors	68	(1)
Measurement of Protein Solutions	69	(5)
The Biuret and Lowry Assays	70	(2)
The Bradford Assay	72	(1)
The BCA Assay	72	(1)
The Spectrophotometric Assay	73	(1)
Measurement of Nucleic Acid Solutions	74	(2)
The Spectrophotometric Assay	74	(1)
Other Assays for Nucleic Acids	74	(2)
Techniques for Sample Preparation	76	(7)
Dialysis	76	(1)
Ultrafiltration	77	(4)
Lyophilization and Centrifugal Vacuum Concentration	81	(2)
Radioisotopes in Biochemistry	83	(18)
Origin and Properties of Radioactivity	84	(4)
Detection and Measurement of Radioactivity	88	(7)
Radioisotopes and Safety	95	(1)
Study Problems	95	(2)
Further Reading	97	(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