Fundamental Laboratory Approaches for Biochemistry and Biotechnology

by Alexander J. Ninfa; David P. Ballou

• ISBN13: 9781891786006
• ISBN10: 1891786008
• Format: Paperback
• Copyright: 1998-04-01
• Publisher: Wiley

Summary

An excellent biochemistry laboratory text for advanced undergraduate and first year graduate students in biochemistry and other life sciences, this text provides a logical framework for training students how to approach research problems and conduct and evaluate scientific research. Each chapter provides extensive background on the principles underlying methods used to research, followed by experiments designed to illustrate those principles.

Author Biography

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.

Table of Contents

Preface	xi
Basic Practices and Techniques in the Biochemistry Laboratory	1	(52)
Laboratory Safety	1	(2)
Laboratory Safety Equipment	3	(1)
Safe Use of Laboratory Reagents	4	(1)
Philosophy and Design of Experiments	5	(5)
Ethics in Science	10	(3)
Presentation and Analysis of Data	13	(3)
Basic Procedures: Measurement of Weights, Volumes, and pH	16	(7)
Various Instruments Used	23	(6)
Other General Techniques	29	(5)
Sample Calculations	34	(2)
Buffers and pH	36	(8)
Keeping a Laboratory Notebook	44	(2)
Laboratory Reports	46	(3)
Calculating Titration Curves for Polyprotic Acids and Other Multiple Binding Site Receptors	49	(2)
Equipment Used in this Book	51	(2)
Spectrophotometry	53	(24)
Introduction	53	(4)
Design and Properties of Spectrophotometers	57	(3)
Effects of Spectral Bandpass and Stray Light	60	(2)
Split Beam Recording Spectrophotometers	62	(1)
Chromogenic Reactions Used for Analysis	62	(3)
Other Forms of Spectroscopy	65	(5)
Experiments 2--1 to 2--4	70	(5)
Reagents Needed for Chapter 2	75	(2)
Quantification of Protein Concentration	77	(12)
Purposes of Protein Quantification	77	(1)
Common Procedures for Protein Quantification	78	(1)
Colorimetric Procedures for Quantification of Proteins	79	(5)
Experiment 3--1	84	(4)
Reagents Needed for Chapter 3	88	(1)
Chromatography	89	(36)
Introduction	89	(4)
Gel-filtration (Size Exclusion or Gel Permeation) Chromatography	93	(7)
Affinity Chromatography	100	(8)
Ion-Exchange Chromatography	108	(5)
Hydrophobic-Interaction Chromatography	113	(4)
Experiments 4--1 to 4--2	117	(7)
Reagents Needed for Chapter 4	124	(1)
Gel Electrophoresis of Proteins	125	(32)
Process of Electrophoresis	125	(2)
Polyacrylamide Gels	127	(2)
SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE) of Proteins	129	(6)
Detection of Proteins in SDS-Polyacrylamide Gels	135	(1)
Applications of SDS-PAGE	136	(4)
Simple (Nondenaturing, Native) Gel Electrophoresis	140	(7)
Experiments 5--1 to 5--2	147	(8)
Reagents Needed for Chapter 5	155	(2)
Overview of Protein Purification	157	(18)
Introduction	157	(1)
Why Do You Want This Protein?	158	(1)
Development of a Suitable Assay Procedure	159	(2)
Time, Temperature, and Yield	161	(1)
Selection of the Best Source Material	161	(2)
Solubilization of the Protein	163	(2)
Initial Steps of Purification	165	(2)
Developing a Series of High-resolution Chromatographic Steps	167	(2)
Methods Used to Change Buffer and Concentrate Protein Samples	169	(1)
A Logical Series of Steps	170	(1)
Storage of the Purified Protein	170	(1)
Protein Purification Table	171	(4)
Isolation and Characterization of the Enzyme Alkaline Phosphatase from Eschericchia Coli	175	(24)
Objectives	175	(1)
Introduction and Basic Principles	175	(4)
Experiment 7--1	179	(11)
Characterization of Purified Alkaline Phosphatase	190	(1)
Assay of Alkaline Phosphatase	191	(5)
Reagents and Equipment Needed for Chapter 7	196	(3)
Enzyme Kinetics	199	(20)
Why Use Steady-state Kinetics?	199	(1)
Steady-state Kinetics Principles	200	(2)
Significance of Km and Vmax	202	(2)
Graphical Analysis	204	(2)
Competitive, ``Noncompetitive,'' and ``Uncompetitive'' Inhibitors	206	(8)
Experiments 8--1 to 8--2	214	(4)
Materials Needed for Chapter 8	218	(1)
Enzymatic Methods of Analysis	219	(28)
Enzymatic Analysis of Substrates	219	(1)
Assays for Enzymatic Activity	220	(1)
Practical Considerations	221	(1)
Coupled Assays	222	(1)
Experiments with Pyridine Nucleotide-Requiring Enzymes	223	(4)
Experiments 9--1 to 9--4	227	(12)
Materials Needed for Chapter 9	239	(3)
Sample Calculations	242	(5)
Ligand Binding	247	(30)
Ligand Binding is the Key to Most Biological Processes	247	(2)
Analysis of Ligand Binding at Equilibrium	249	(3)
Digression on Regression	252	(3)
Effects of the Concentration of L and R	255	(1)
Effects of Two Sites or Cooperativity	256	(5)
Analysis of the Kinetics of Ligand Binding	261	(3)
Methods Used To Study Receptor-Ligand Interactions	264	(8)
Experiment 10--1	272	(3)
Reagents Needed for Chapter 10	275	(2)
Recombinant DNA Techniques	277	(36)
Introduction	277	(1)
Overview of the Strategy of Recombinant DNA Techniques	278	(2)
Cutting and Splicing DNA	280	(3)
Gel Electrophoresis of DNA	283	(3)
Introducing DNA into Cells	286	(1)
Identifying Transformed Cells	287	(4)
There Are Many Useful Tools for Recombinant DNA Experiments	291	(4)
Applications of Recombinant DNA Technology	295	(5)
Experiments 11--1 to 11--4	300	(10)
Reagents Needed for Chapter 11	310	(3)
Polymerase Chain Reaction (PCR) Technology	313	(24)
Introduction	313	(1)
Principle of the PCR Method	313	(3)
Applications of PCR	316	(8)
Experiments 12--1 to 12--5	324	(11)
Materials and Reagents Needed for Chapter 12	335	(2)
Using the Personal Computer and the Internet for Biochemical Research	337	(24)
Introduction to the World Wide Web (the Internet)	337	(7)
Information on the Internet Useful to Biochemists	344	(8)
Using RasMol to View Molecular Structures	352	(2)
Other Useful Information on the Internet	354	(2)
Homework Assignment	356	(4)
Materials Needed for Chapter 13	360	(1)
Appendix 1: Safety Checklist	361	(1)
Appendix 2: An Example of an MSDS	362	(3)
Index	365