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ID:40960716
大小:4.48 MB
页数:524页
时间:2019-08-12
《Protein Purification Protocols 2nd ed》由会员上传分享,免费在线阅读,更多相关内容在学术论文-天天文库。
1、MethodsinMolecularBiologyMethodsinMolecularBiologyTMTMVOLUME244ProteinProteinPurificationPurificationProtocolsProtocolsSecondEditionSecondEditionEditedbyEditedbyPaulCutlerPaulCutler1GeneralStrategiesShawnDoonanandPaulCutler1.DefiningtheProblemThechaptersthatfollowinthisvolumegivedetailedinstructionso
2、nhowtousethevariousmethodsthatareavailableforpurificationofproteins.Thequestionarises,how-ever,ofwhichofthesemethodstouseandinwhichordertousethemtoachievepu-rificationinanyparticularcase;thatis,thepurificationproblemmustbeclearlydefined.Whatfollowsoutlinesthesortsofquestionthatneedtobeaskedaspartofthatd
3、efini-tionandhowtheanswersaffecttheapproachthatmightbetakentodevelopingapu-rificationschedule.Itshouldbenotedherethatthediscussionconcentratesmainlyonlaboratory-scaleisolationofproteins.Specialcasesofpurificationoftherapeuticpro-teinsandisolationatindustrialscalearecoveredinChapters43and44(1–5).1.1.How
4、MuchDoINeed?Theanswertothisquestiondependsonthepurposeforwhichtheproteinisrequired.Forexample,tocarryoutafullchemicalandphysicalanalysisofaproteinmayrequireseveralhundredsofmilligramsofpurifiedmaterial,whereasakineticanalysisofthere-actioncatalyzedbyanenzymecouldperhapsbedonewithafewmilligramsandless
5、than1mgwouldberequiredtoraiseapolyclonalantibody.Attheextremeendofthescale,iftheobjectiveistoobtainlimitedsequenceinformationfromtheN-terminusofaproteinasapreliminarytothedesignofanoligonucleotideprobeforclonescreening,thenusingmodernmicrosequencingtechniques,afewmicrogramswillbesufficient.Inthefield
6、ofproteomics,previouslyanalyticaltechniqueshavebecomepreparativewithmassspec-trometrycommonplaceforsensitiveproteincharacterizationfromspotsongels.Chap-ters36and40–42describethesemethodologies.Thesedifferentrequirementsforquan-titymaywelldictatethesourceoftheproteinchosen(seeSubheading1.4.)andwillce
7、rtainlyinfluencetheapproachtopurification.Purificationoflargequantitiesofproteinrequiresuseoftechniques,atleastintheearlystages,thathaveahighcapacitybutlowresolvingpower,suchasfractionalprecipitationwith
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