Proteinpurification;actin

OverviewACTINThemostabundantmuscleandnon-musclecytoskeletalprotein.MW42kDa,374/375aminoacids;variousisoforms;structureresolvedat2.8Angstrom.[seealsoprotocolID207,208,209]

Material

1.Source:rabbitskeletalmuscle2.Equipment:•mincer•glass-Teflon-homogenizerwithpestle•sephacrylS-300column(5x100cm)•electrophoreticgels•centrifuge•sterilecheesecloth3.Chemicals:KCl,EDTA,acetone,TrismaBase,CaCl2,MgCl2,ATP,DTT,NaN3;columnmaterial:sephacrylS-3004.Haveready:bufferG:2mMTris-HCl,0.2mMCaCl2,0.2mMATP,0.2mMDTT,0.05%NaN3,pH8.0(15liter)

Procedure

FLOWCHART1.take~350gramrabbitskeletalmuscle2.extractat+4oCwith0.1MKClandfilter3.(takeresidue)extractat+4oCwith50mMNaHCO3andfilter4.(takeresidue)extractat+4oCwith1mMEDTA,pH7.0andfilter5.(takeresidue)extractat+4oCwithH2O(2x)andfilter6.(takeresidue)extractat+20-25oCwithacetone(5x)andfilter7.(usemuscleacetonepowder)extractwithbufferGat0oC;20ml/7gramacetonepowder;centrifugeat35,000xgfor30min.8.(usecombinedfiltrate)filterthroughsterilecheeseclothat+4oC9.(usesupernatant)add50mMKCl,2mMMgCl2,0.5mMATPforpolymerization;incubatefor30min;stirfor30min;adjustto0.8MKCl;centrifugeat100,000xgfor2h10.(takemuscleF-actin;notsupernatant)homogenizein300mlbufferGat+4oC;dialyzesUSPensionagainstbufferGfor48h;centrifugeat100,000xgfor2h11.(takemuscleG-actin;notpellet)chromatographonsephacrylS-300columnat+4oC12.poolG-actinfractionsat+4oC(~10mgG-actin/gramacetonepowder)

Reference?Sheterline,P.ActinProfileVol.1,1(1994)AcademicPress,London,UK.?Kabsch,W.,Mannherz,H.G.,Suck,D.,Pai,E.F.andHolmes,K.C.Nature(1990)347:37-44.?Goldmann,W.H.Biochem.Biophys.Res.Commun.(2000)271:553-557.

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