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Principals and Techiniques of Biochemistry and Molecular Biology-7th PDF

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Principles and Techniques of Biochemistry and Molecular Biology Seventh edition EDITED BY KEITH WILSON AND JOHN WALKER Thisneweditionofthebestsellingtextbookintegratesthetheoreticalprinciplesandexperimental techniquescommontoallundergraduatecoursesinthebio-andmedicalsciences.Threeofthe 16chaptershavenewauthorsandhavebeentotallyrewritten.Theothershavebeenupdatedand extendedtoreflectdevelopmentsintheirfieldexemplifiedbyanewsectiononstemcells.Twonew chaptershavebeenadded.Oneonclinicalbiochemistrydiscussestheprinciplesunderlyingthediagnosis andmanagementofcommonbiochemicaldisorders.Thesecondoneondrugdiscoveryanddevelopment illustrateshowtheprinciplesandtechniquescoveredinthebookarefundamentaltothedesignand developmentofnewdrugs.In-textworkedexamplesareagainusedtoenhancestudentunderstanding ofeachtopicandcasestudiesareselectivelyusedtoillustrateimportantexamples.Experimental design,qualityassuranceandthestatisticalanalysisofquantitativedataareemphasisedthroughout thebook. • Motivatesstudentsbyincludingcutting-edgetopicsandtechniques,suchasdrugdiscovery, aswellasthemethodstheywillencounterintheirownlabclasses • Promotesproblemsolvingbysettingstudentsachallengeandthenguidingthemthrough thesolution • Integratestheoryandpractisetoensurestudentsunderstandwhyandhoweachtechnique isused. KEITH WILSON isProfessorEmeritusofPharmacologicalBiochemistryandformerHeadofthe DepartmentofBiosciences,DeanoftheFacultyofNaturalSciences,andDirectorofResearchatthe UniversityofHertfordshire. JOHN WALKER isProfessorEmeritusandformerHeadoftheSchoolofLifeSciencesatthe UniversityofHertfordshire. Coverillustration Mainimage Electrophoresisgelshowingrecombinantprotein.Photographer:J.C.Revy.Courtesyof SciencePhotoLibrary. Topinset TranscriptionfactorandDNAmolecule.Courtesyof:LagunaDesign/SciencePhotoLibrary. Secondinset Microtubes,pipettor(pipette)tip&DNAsequence.CourtesyofTekImage/Science PhotoLibrary. Thirdinset Stemcellculture,lightmicrograph.Photographer:PhilippePlailly.CourtesyofScience PhotoLibrary. Fourthinset Embryonicstemcells.CourtesyofSciencePhotoLibrary. Bottominset Herceptinbreastcancerdrug,molecularmodel.Photographer:TimEvans.Courtesyof SciencePhotoLibrary. Principles and Techniques of Biochemistry and Molecular Biology Seventh edition Edited by KEITH WILSON AND JOHN WALKER CAMBRIDGEUNIVERSITYPRESS Cambridge,NewYork,Melbourne,Madrid,CapeTown,Singapore, Sa˜oPaulo,Delhi,Dubai,Tokyo CambridgeUniversityPress TheEdinburghBuilding,CambridgeCB28RU,UK PublishedintheUnitedStatesofAmericaby CambridgeUniversityPress,NewYork www.cambridge.org Informationonthistitle:www.cambridge.org/9780521516358 Firstandsecondeditions#BryanWilliamsandKeithWilson1975,1981 Thirdedition#KeithWilsonandKennethH.Goulding1986 Fourthedition#CambridgeUniversityPress1993 Fifthedition#CambridgeUniversityPress2000 Sixthedition#CambridgeUniversityPress2005 Seventhedition#CambridgeUniversityPress2010 Thispublicationisincopyright.Subjecttostatutoryexception andtotheprovisionsofrelevantcollectivelicensingagreements, noreproductionofanypartmaytakeplacewithout thewrittenpermissionofCambridgeUniversityPress. FirstpublishedbyEdwardArnold1975asABiologist’sGuidetoPrinciplesandTechniquesofPractical Biochemistry Secondedition1981;Thirdedition1986 ThirdeditionfirstpublishedbyCambridgeUniversityPress1992;Reprinted1993 FourtheditionpublishedbyCambridgeUniversityPress1994asPrinciplesandTechniquesofPractical Biochemistry;Reprinted1995,1997;Fifthedition2000 SixtheditionfirstpublishedbyCambridgeUniversityPress2005asPrinciplesandTechniquesofBiochemistry andMolecularBiology;Reprinted2006,2007 SeventheditionfirstpublishedbyCambridgeUniversityPress2010 PrintedintheUnitedKingdomattheUniversityPress,Cambridge AcataloguerecordforthispublicationisavailablefromtheBritishLibrary LibraryofCongressCataloging-in-PublicationData Principlesandtechniquesofbiochemistryandmolecularbiology/editedbyKeithWilson, JohnWalker.–7thed. p. cm. ISBN978-0-521-51635-8(hardback)–ISBN978-0-521-73167-6(pbk.) 1. Biochemistry–Textbooks. 2. Molecularbiology–Textbooks. I. Wilson,Keith,1936– II. Walker, JohnM.,1948– III. Title. QP519.7.P752009 6120.015–dc22 2009043277 ISBN978-0-521-51635-8Hardback ISBN978-0-521-73167-6Paperback CambridgeUniversityPresshasnoresponsibilityforthepersistenceor accuracyofURLsforexternalorthird-partyinternetwebsitesreferredto inthispublication,anddoesnotguaranteethatanycontentonsuch websitesis,orwillremain,accurateorappropriate. CONTENTS Prefacetotheseventhedition pagexi Listofcontributors xiii Listofabbreviations xv 1 Basic principles 1 K. WILSON 1.1 Biochemicalandmolecularbiologystudies 1 1.2 Unitsofmeasurement 3 1.3 Weakelectrolytes 6 1.4 Quantitativebiochemicalmeasurements 16 1.5 Safetyinthelaboratory 35 1.6 Suggestionsforfurtherreading 37 2 Cell culture techniques 38 A.R. BAYDOUN 2.1 Introduction 38 2.2 Thecellculturelaboratoryandequipment 39 2.3 Safetyconsiderationsincellculture 43 2.4 Aseptictechniquesandgoodcellculturepractice 44 2.5 Typesofanimalcell,characteristicsandmaintenanceinculture 49 2.6 Stemcellculture 61 2.7 Bacterialcellculture 68 2.8 Potentialuseofcellcultures 71 2.9 Suggestionsforfurtherreading 72 3 Centrifugation 73 K. OHLENDIECK 3.1 Introduction 73 3.2 Basicprinciplesofsedimentation 74 3.3 Types,careandsafetyaspectsofcentrifuges 79 3.4 Preparativecentrifugation 86 3.5 Analyticalcentrifugation 95 3.6 Suggestionsforfurtherreading 99 v vi Contents 4 Microscopy 100 S. W. PADDOCK 4.1 Introduction 100 4.2 Thelightmicroscope 103 4.3 Opticalsectioning 116 4.4 Imaginglivingcellsandtissues 123 4.5 Measuringcellulardynamics 126 4.6 Theelectronmicroscope(EM) 129 4.7 Imagearchiving 133 4.8 Suggestionsforfurtherreading 136 5 Molecular biology, bioinformatics and basic techniques 138 R. RAPLEY 5.1 Introduction 138 5.2 Structureofnucleicacids 139 5.3 Genesandgenomecomplexity 145 5.4 Locationandpackagingofnucleicacids 149 5.5 Functionsofnucleicacids 152 5.6 Themanipulationofnucleicacids–basictoolsandtechniques 162 5.7 Isolationandseparationofnucleicacids 164 5.8 Molecularbiologyandbioinformatics 170 5.9 Molecularanalysisofnucleicacidsequences 171 5.10 Thepolymerasechainreaction(PCR) 178 5.11 NucleotidesequencingofDNA 187 5.12 Suggestionsforfurtherreading 194 6 Recombinant DNA and genetic analysis 195 R. RAPLEY 6.1 Introduction 195 6.2 Constructinggenelibraries 196 6.3 Cloningvectors 206 6.4 Hybridisationandgeneprobes 223 6.5 Screeninggenelibraries 225 6.6 Applicationsofgenecloning 229 6.7 Expressionofforeigngenes 234 6.8 Analysinggenesandgeneexpression 240 6.9 Analysingwholegenomes 254 6.10 Pharmacogenomics 259 6.11 Molecularbiotechnologyandapplications 260 6.12 Suggestionsforfurtherreading 262 7 Immunochemical techniques 263 R. BURNS 7.1 Introduction 263 7.2 Makingantibodies 273 vii Contents 7.3 Immunoassayformats 283 7.4 Immunomicroscopy 291 7.5 Lateralflowdevices 291 7.6 Epitopemapping 292 7.7 Immunoblotting 293 7.8 Fluorescentactivatedcellsorting(FACS) 293 7.9 Cellandtissuestainingtechniques 294 7.10 Immunocapturepolymerasechainreaction(PCR) 295 7.11 Immunoaffinitychromatography(IAC) 295 7.12 Antibody-basedbiosensors 296 7.13 Therapeuticantibodies 297 7.14 Thefutureusesofantibodytechnology 299 7.15 Suggestionsforfurtherreading 299 8 Protein structure, purification, characterisation and function analysis 300 J. WALKER 8.1 Ionicpropertiesofaminoacidsandproteins 300 8.2 Proteinstructure 304 8.3 Proteinpurification 307 8.4 Proteinstructuredetermination 328 8.5 Proteomicsandproteinfunction 340 8.6 Suggestionsforfurtherreading 351 9 Mass spectrometric techniques 352 A. AITKEN 9.1 Introduction 352 9.2 Ionisation 354 9.3 Massanalysers 359 9.4 Detectors 377 9.5 Structuralinformationbytandemmassspectrometry 379 9.6 Analysingproteincomplexes 390 9.7 Computinganddatabaseanalysis 394 9.8 Suggestionsforfurtherreading 397 10 Electrophoretic techniques 399 J. WALKER 10.1 Generalprinciples 399 10.2 Supportmedia 403 10.3 Electrophoresisofproteins 407 10.4 Electrophoresisofnucleicacids 422 10.5 Capillaryelectrophoresis 427 10.6 Microchipelectrophoresis 431 10.7 Suggestionsforfurtherreading 432 viii Contents 11 Chromatographic techniques 433 K. WILSON 11.1 Principlesofchromatography 433 11.2 Chromatographicperformanceparameters 435 11.3 High-performanceliquidchromatography 446 11.4 Adsorptionchromatography 453 11.5 Partitionchromatography 455 11.6 Ion-exchangechromatography 459 11.7 Molecular(size)exclusionchromatography 462 11.8 Affinitychromatography 465 11.9 Gaschromatography 470 11.10 Suggestionsforfurtherreading 476 12 Spectroscopic techniques: I Spectrophotometric techniques 477 A. HOFMANN 12.1 Introduction 477 12.2 Ultravioletandvisiblelightspectroscopy 482 12.3 Fluorescencespectroscopy 493 12.4 Luminometry 507 12.5 Circulardichroismspectroscopy 509 12.6 Lightscattering 514 12.7 Atomicspectroscopy 516 12.8 Suggestionsforfurtherreading 519 13 Spectroscopic techniques: II Structure and interactions 522 A. HOFMANN 13.1 Introduction 522 13.2 InfraredandRamanspectroscopy 523 13.3 Surfaceplasmonresonance 527 13.4 Electronparamagneticresonance 530 13.5 Nuclearmagneticresonance 536 13.6 X-raydiffraction 546 13.7 Small-anglescattering 549 13.8 Suggestionsforfurtherreading 551 14 Radioisotope techniques 553 R.J. SLATER 14.1 Whyusearadioisotope? 553 14.2 Thenatureofradioactivity 554 14.3 Detectionandmeasurementofradioactivity 561 14.4 Otherpracticalaspectsofcountingofradioactivityandanalysisofdata 573 14.5 Safetyaspects 577 14.6 Suggestionsforfurtherreading 580 ix Contents 15 Enzymes 581 K. WILSON 15.1 Characteristicsandnomenclature 581 15.2 Enzymesteady-statekinetics 584 15.3 Analyticalmethodsforthestudyofenzymereactions 602 15.4 Enzymeactivesitesandcatalyticmechanisms 611 15.5 Controlofenzymeactivity 615 15.6 Suggestionsforfurtherreading 624 16 Principles of clinical biochemistry 625 J. FYFFE AND K. WILSON 16.1 Principlesofclinicalbiochemicalanalysis 625 16.2 Clinicalmeasurementsandqualitycontrol 629 16.3 Examplesofbiochemicalaidstoclinicaldiagnosis 640 16.4 Suggestionsforfurtherreading 658 16.5 Acknowledgements 659 17 Cell membrane receptors and cell signalling 660 K. WILSON 17.1 Receptorsforcellsignalling 660 17.2 Quantitativeaspectsofreceptor–ligandbinding 663 17.3 Ligand-bindingandcell-signallingstudies 680 17.4 Mechanismsofsignaltransduction 685 17.5 Receptortrafficking 703 17.6 Suggestionsforfurtherreading 707 18 Drug discovery and development 709 K. WILSON 18.1 Humandiseaseanddrugtherapy 709 18.2 Drugdiscovery 718 18.3 Drugdevelopment 727 18.4 Suggestionsforfurtherreading 734 Index 736 Thecolourfiguresectionisbetweenpages128and129

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