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1、沉淀蛋白质的常用方法（TCA、乙醇、丙酮沉淀蛋白操作步骤）TCA-DOCForprecipitationofverylowproteinconcentration1)Toonevolumeofproteinsolution,add1/100vol.of2%DOC(Nadeoxycholate,detergent).2)Vortexandletsitfor30minat4oC.3)Add1/10ofTrichloroaceticacid(TCA)100%vortexandletsitONat4oC(preparationof100%TCA:454mlH2O/kgTCA.Maintainindar
2、kbottleat4oC.Becareful,usegloves!).4)Spin15min4oCinmicrofugeatmaximumspeed(15000g).Carefullydischargesupernatantandretainthepellet:drytubebyinversionontissuepaper(pelletmaybedifficulttosee).OPTION:Washpellettwicewithonevolumeofcoldacetone(acetonekeepat20oC).Vortexandrepelletsamples5minatfullspeedbet
3、weenwashes.5)Drysamplesundervaccum(speedvac)ordryair.ForPAGE-SDS,resuspendsamplesinaminimalvolumeofsamplebuffer.(ThepresenceofsomeTCAcangiveayellowcolourasaconsequenceoftheacidificationofthesamplebuffertitratewith1NNaOHor1MTrisHClpH8.5toobtainthenormalbluesamplebuffercolour.)NormalTCAToeliminateTCAs
4、olubleinterferencesandproteinconcentration1)ToasampleofproteinsolutionaddTrichloroaceticacid(TCA)100%toget13%finalconcentration.Mixandkeep5min20oCandthen15min4oC;orlongertimeat4oCwithoutthe20oCstepforlowerproteinconcentration.Suggestion: leaveONiftheproteinconcentrationisverylow.(preparationof100%TC
5、A:454mlH2O/kgTCA.Maintainindarkbottleat4oC.Becareful,usegloves!).2)Spin15min4oCinmicrofugeatmaximumspeed(15000g).Carefullydischargesupernatantandretainthepellet:drytubebyinversionontissuepaper(pelletmaybedifficulttosee).3)ForPAGE-SDS,resuspendsamplesinaminimalvolumeofsamplebuffer.(ThepresenceofsomeT
6、CAcangiveayellowcolourasaconsequenceoftheacidificationofthesamplebuffertitratewith1NNaOHor1MTrisHClpH8.5toobtainthenormalbluesamplebuffercolour.)AcetonePrecipitationToeliminateacetonesolubleinterferencesandproteinconcentration1)Addto1volumeofproteinsolution4volumesofcoldacetone.Mixandkeepatleast20mi
7、n20oC.(Suggestion:leaveONiftheproteinconcentrationisverylow).2)Spin15min4oCinmicrofugeatmaximumspeed(15000g).Carefullydischargesupernatantandretainthepellet:drytubebyinversionontissuepaper(pelletmaybedifficulttosee).3)Drysamplesundervaccum(speed-vac)ordryairtoeliminateanyacetoneresidue(smelltubes).F
8、orPAGE-SDS,resuspendsamplesinaminimalvolumeofsamplebuffer.EthanolPrecipitationUsefulmethodtoconcentrateproteinsandremovalofGuanidineHydrochloridebeforePAGE-SDS1)Addto1volumeofproteinsolution9volumesofcoldEthanol100%.Mixandkeepatleast10min.at20oC.(Suggestion:leaveON).2)Spin15min4oCinmicrocentrifugeat
9、maximumspeed(15000g).Carefullydischargesupernatantandretainthepellet:drytubebyinversionontissuepaper(pelletmaybedifficulttosee).3)Washpelletwith90%coldethanol(keepat20oC).Vortexandrepelletsamples5minatfullspeed.4)Drysamplesundervaccum(speedvac)ordryairtoeliminateanyethanolresidue(smelltubes).ForPAGE
10、-SDS,resuspendsamplesinaminimalvolumeofsamplebuffer.TCA-DOC/AcetoneUsefulmethodtoconcentrateproteinsandremoveacetoneandTCAsolubleinterferences1.Toonevolumeofproteinsolutionadd2%Nadeoxycholate(DOC)to0.02%final(for100lsample,add1l2%DOC).2.Mixandkeepatroomtemperatureforatleast15min.3.100%trichloroaceti
11、cacid(TCA)toget10%finalconcentration(preparationof100%TCA:454mlH2O/kgTCA.Maintainindarkbottleat4oC.Becareful,usegloves!).4.Mixandkeepatroomtemperatureforatleast1hour.5.Spinat4oCfor10min,removesupernatantandretainthepellet.Drytubebyinversionontissuepaper.6.Add200loficecoldacetonetoTCApellet.7.Mixandk
12、eeponiceforatleast15min.8.Spinat4oCfor10mininmicrocentrifugeatmaximumspeed.9.Removesupernatantasbefore(5),dryairpellettoeliminateanyacetoneresidue(smelltubes).ForPAGE-SDS,resuspendsamplesinaminimalvolumeofsamplebuffer.10.(ThepresenceofsomeTCAcangiveayellowcolourasaconsequenceoftheacidificationofthes
13、amplebuffertitratewith1NNaOHor1MTrisHClpH8.5toobtainthenormalbluesamplebuffercolour.)AcidifiedAcetone/MethanolUsefulmethodtoremoveacetoneandmethanolsolubleinterferenceslikeSDSbeforeIEF1)Prepareacidifiedacetone:120mlacetone+10lHCl(1mMfinalconcentration).2)Prepareprecipitationreagent:Mixequalvolumesofacidifiedacetoneandmethanolandkeepat-20oC.3)Toonevolumeofproteinsolutionadd4volumesofcoldprecipitationreagent.MixandkeepONat-20oC.4)Spin15mi
