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troponin蛋白纯化 Protein purification: troponins

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Overview

TROPONINSThecalcium-dependentregulatoryproteincomplexlocatedonthethinactinfilamentsofmusclecomprisesofTnC(17.8kDa),TnI(20.8kDa),andTnT(30.5kDa).Theseproteinsareinvolvedinthekeyregulatorymechanismformusclecontration.

Material

Source:skeletalmuscleetherpowderEquipment:•centrifuge•CibaronBlue-sephacrylcolumn(5x50cm)•DEAE-sephadexA-50column(2.5x25cm)•CM-sephadexC-50-120column(2.5x25cm)•SDS-PageChemicals:Tris-HCl,KCl,CaCl2,EDTA,DTT,1NHCl,1NKOH,imidazole,urea,citrate,NaN3Haveready:extractionsolution:1MKCl,25mMTris-HCl,0.1mMCaCl2,0.1mMDTT,pH8.0dialyzingbuffer1:10mMimidazole,50mMKCl,0.1mMCaCl2,0.1mMDTT,0.02%NaN3,pH7.0dialyzingbuffer2:2mMTris-HCl,0.1mMCaCl2,pH8.0dialyzingbuffer3:6Murea,50mMTris-HCl,1mMEDTA,0.1mMDTT,pH8.0dialyzingbuffer4:6Murea,50mMcitrate,1mMEDTA,0.1mMDTT,pH6.0

Procedure

FLOWCHART(allproceduresarecarriedoutat4oC)1.takeskeletalmuscleetherpowder(~150gram)2.extractovernightwithextractionsolution(15:1,v/w);centrifugeat10.9xgfor10min3.(takepellet)reextract,add1MKCltocentrifugebottles(7.5:1,v/w);centrifugeat10.9xgfor10min4.(takesupernatant)lowerpHto4.6byadding1NHCl,removeprecipitate(tropomyosin)bycentrifugationat7020xgfor10min5.(takesupernatant)adjustpHto8.0byadding1NKOH;make40%(NH4)2SO4(230gram/liter);centrifugeat10.9xgfor10min6.(takepellet)resUSPendinminimalvolumeofdialyzingbuffer1;dialyzeagainst4x1liter;clarifyat31,200xgfor20min7.applytobluesephacrylcolumn8.equilibratewithdialyzingbuffer;washwiththesamebuffer(1liter)andeluteinstepsof0.1,0.2,0.3,0.4,0.5,and0.6MKCl,1litereach;collect25mlfractions>200/<220ml;storefrozenat-80oCorlyophilizeat-20oC-->troponin0.6gramtotalForTnC,TnTaBDTnI:1.(takepelletobtainedinstep5)resuspendinminimalvolofdialyzingbuffer2;dialyzeagainst16literofdialyzingbuffer2;centrifugeat31,300xgfor15min2.(takesupernatant)dialyzeagainstdialyzingbuffer3(2x2liter)(ureasettlesdowninthebag!)3.applytoDEAE-sephadexA50column4.equilibratewithdialyzingbuffer3;elutewithlineargrADIent0-0.5MKCl(2x500ml);collect6mlfractionsToobtainpureTnI,TnCandTnT:dialyzeagainstdialyzingbuffer4(1liter)andloadontoCM-sephadexcolumn;elutewithlineargradient0-0.6MNaCl(2x0.5liter);collect6mlfractions-->TnI,TnC,TnT

Reference

•Potter,J.D.(1982)MethodsEnzymol.85,241-263.•Goldmann,W.H.(2000)Biochem.Biophys.Res.Comm.276,1225-1228.

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troponin蛋白纯化&nbsp;Protein&nbsp;purification:&nbsp;troponins

troponin蛋白纯化 Protein purification: troponins