Principles and Techniques of Practical Biochemistry

by Edited by Keith Wilson , John Walker

• ISBN13: 9780521658737
• ISBN10: 052165873X
• Edition: 5th
• Format: Paperback
• Copyright: 2000-04-13
• Publisher: Cambridge University Press

Summary

In this new, 5th edition of a highly popular text, undergraduate students are introduced to all the basic experimental techniques routinely used in practical biochemistry today. Most attention is given to techniques students will encounter in their practical classes, with the principles and theories behind them explained in detail to aid understanding. As a further aid to students, essential calculations and worked answers appear at the end of each chapter. 'Key terms to understand' are also included to help students thoroughly review each topic. No contemporary book on modern biochemical techniques would be complete without chapters on molecular biology, recombinant DNA technology, genetic analysis and biomolecular interactions, and these topics have been extensively covered in this new edition. The book is essential reading for all bioscience undergraduate students and pre-clinical medical students for whom practical biochemistry, molecular biology and immunology form part of the syllabus.

Table of Contents

Preface to the fifth edition	xiii
List of contributors	xvi
List of abbreviations	xviii
General principles of biochemical investigations
I. Simpkins
The nature of biochemistry	1	(3)
Bioenergetics	4	(17)
Methods for investigating metabolism	21	(7)
Practical considerations	28	(14)
In vivo models	42	(2)
In vitro models	44	(22)
Microscopy	66	(9)
Key terms	75	(1)
Calculations	76	(2)
Suggestions for further reading	78	(2)
Molecular biology and basic techniques	80	(58)
R. Rapley
Introduction	80	(1)
Components and primary structure of nucleic acids	80	(7)
Genes and genome complexity	87	(3)
The nature of the genetic code	90	(1)
Cellular location of nucleic acids	90	(3)
The cellular functions of DNA	93	(10)
The manipulation of nucleic acids: basic tools and techniques	103	(2)
Isolation and separation of nucleic acids	105	(5)
Restriction mapping of DNA fragments	110	(1)
Nucleic acid blotting methods	111	(2)
Gene probe derivation	113	(1)
Labelling DNA gene probe molecules	114	(2)
The polymerase chain reaction	116	(9)
Nucleotide sequencing of DNA	125	(6)
Bioinformatics and the Internet	131	(3)
Key terms	134	(1)
Calculations	135	(1)
Suggestions for further reading	136	(2)
Molecular cloning and gene analysis	138	(68)
R. Rapley
Introduction	138	(1)
Constructing gene libraries	138	(10)
Cloning vectors	148	(19)
Hybridisation and gene probes	167	(1)
Screening gene libraries	167	(4)
Applications of gene cloning	171	(7)
Expression of foreign genes	178	(6)
Analysing genes and gene expression	184	(12)
Analysing whole genomes	196	(6)
Molecular biotechnology and applications	202	(2)
Key terms	204	(1)
Suggestions for further reading	205	(1)
Immunochemical techniques	206	(57)
R. Thorpe
S. Thorpe
Introduction	206	(5)
Production of antibodies	211	(11)
Purification and fragmentation of immunoglobulins	222	(7)
Immunoprecipitation	229	(5)
Labelling antibodies	234	(6)
Immunoblotting	240	(4)
Immunoassays	244	(10)
Immunohisto/cytochemistry	254	(6)
Affinity and avidity	260	(1)
Immunochemical use of surface plasmon resonance	260	(1)
Key terms	261	(1)
Calculation	262	(1)
Suggestions for further reading	262	(1)
Centrifugation techniques	263	(49)
A. Griffiths
Introduction	263	(1)
Basic principles of sedimentation	264	(7)
Centrifuges and their uses	271	(5)
Design and care of preparative rotors	276	(8)
Sample containers	284	(1)
Separation methods in preparative ultracentrifuges	285	(5)
Performing density gradient separations	290	(6)
Selection, efficiency and applications of preparative rotors	296	(4)
Analysis of subcellular fractions	300	(2)
Some applications of the analytical ultracentrifuge	302	(3)
Safety aspects in the use of centrifuges	305	(1)
Key terms	306	(1)
Calculations	306	(5)
Suggestions for further reading	311	(1)
Protein structure, purification and characterisation	312	(45)
J. Walker
Ionic properties of amino acids and proteins	312	(4)
Protein structure	316	(2)
Protein purification	318	(20)
Protein structure determination	338	(15)
Key terms	353	(1)
Calculations	354	(2)
Suggestions for further reading	356	(1)
Biomolecular interactions: I Enzymes	357	(46)
K. Wilson
Receptor--ligand binding	357	(1)
Enzymes: characteristics and nomenclature	358	(1)
Enzyme steady-state kinetics	359	(19)
Enzyme assays	378	(7)
Substrate assays	385	(1)
Enzyme pre-steady-state kinetics	386	(3)
Enzyme active sites and catalytic mechanisms	389	(5)
Immobilised enzymes	394	(2)
Cellular control of metabolic activity	396	(4)
Key terms	400	(1)
Calculations	401	(1)
Suggestions for further reading	402	(1)
Biomolecular interactions: II Cell surface receptors and transporters	403	(50)
K. Wilson
Cell surface receptor classification	403	(3)
Quantitative aspects of receptor-ligand binding	406	(11)
Receptor structures	417	(3)
Mechanisms of signal transduction	420	(10)
Signal amplification	430	(2)
Key terms	432	(1)
Membrane transport processes	432	(1)
Physical diffusion	433	(2)
Facilitated transport	435	(3)
Active transport and ion channels	438	(7)
Receptor-mediated endocytosis	445	(4)
Key terms	449	(1)
Calculations	449	(2)
Suggestions for further reading	451	(2)
Spectroscopic techniques: I Atomic and molecular electronic spectroscopy	453	(45)
D. B. Gordon
Introduction	453	(3)
γ-Ray spectroscopy and γ-ray resonance spectroscopy	456	(2)
X-ray spectroscopy	458	(1)
Ultraviolet and visible light spectroscopy	459	(12)
Spectrofluorimetry	471	(8)
Circular dichroism spectroscopy	479	(3)
Turbidimetry and nephelometry	482	(1)
Luminometry	483	(2)
Atomic spectroscopy	485	(5)
Lasers	490	(1)
Key terms	491	(1)
Calculations	492	(5)
Suggestions for further reading	497	(1)
Spectroscopic techniques: II Vibrational spectroscopy and electron and nuclear spin orientation in magnetic fields	498	(29)
D. B. Gordon
Introduction	498	(1)
Infrared and Raman spectroscopy	498	(3)
Electron spin resonance spectroscopy	501	(7)
Nuclear magnetic resonance spectroscopy	508	(17)
Key terms	525	(1)
Suggestions for further reading	526	(1)
Mass spectrometric techniques	527	(53)
D. B. Gordon
Introduction	527	(1)
The mass spectrometer	527	(2)
Electron impact ionisation	529	(9)
Chemical ionisation	538	(1)
Field ionisation	539	(1)
Ion desorption methods	539	(12)
Ion evaporation methods	551	(4)
Analysers	555	(9)
Detectors	564	(2)
Tandem mass spectrometry	566	(7)
Key terms	573	(1)
Calculations	573	(6)
Suggestions for further reading	579	(1)
Electrophoretic techniques	580	(39)
J. M. Walker
General principles	580	(4)
Support media	584	(4)
Electrophoresis of proteins	588	(19)
Electrophoresis of nucleic acids	607	(5)
Capillary electrophoresis	612	(5)
Key terms	617	(1)
Calculation	618	(1)
Suggestions for further reading	618	(1)
Chromatographic techniques	619	(68)
K. Wilson
Introduction	619	(4)
Chromatography theory and practice	623	(8)
Low pressure column chromatography	631	(6)
High performance liquid chromatography	637	(10)
Adsorption chromatography	647	(2)
Partition chromatography	649	(7)
Ion-exchange chromatography	656	(5)
Molecular exclusion (permeation) chromatography	661	(4)
Affinity chromatography	665	(7)
Gas-liquid chromatography	672	(6)
Thin-layer (planar) chromatography	678	(3)
Selection of a chromatographic system	681	(1)
Key terms	682	(1)
Calculations	683	(3)
Suggestions for further reading	686	(1)
Radioisotope techniques	687	(42)
R. J. Slater
The nature of radioactivity	687	(6)
Detection and measurement of radioactivity	693	(20)
Other practical aspects of counting radioactivity and analysis of data	713	(4)
Inherent advantages and restrictions of radiotracer experiments	717	(1)
Safety aspects	718	(3)
Applications of radioisotopes in the biological sciences	721	(5)
Key terms	726	(1)
Calculations	726	(2)
Suggestions for further reading	728	(1)
Electrochemical techniques	729	(42)
P. K. Robinson
Introduction	729	(5)
Principles of electrochemical techniques	734	(8)
Redox reactions	742	(3)
The pH electrode	745	(3)
Ion-selective and gas-sensing electrodes	748	(2)
The Clark oxygen electrode	750	(7)
Electrochemical detectors for HPLC	757	(3)
Biosensors	760	(8)
Key terms	768	(1)
Calculations	768	(2)
Suggestions for further reading	770	(1)
Index	771