Protein Purification Protocols 2nd ed

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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