沉淀蛋白质的常用方法(TCA、乙醇、丙酮沉淀蛋白操作步骤)

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.Maintainindarkbottleat4oC.Becareful,usegloves!!!).4)Spin15min4oCinmicrofugeatmaximumspeed(15000g).Carefullydischargesupernatantandretainthepellet:drytubebyinversionontissuepaper(pelletmaybedifficulttosee).[OPTION:Washpellettwicewithonevolumeofcoldacetone(acetonekeepat–20oC).Vortexandrepelletsamples5minatfullspeedbetweenwashes].5)Drysamplesundervaccum(speedvac)ordryair.ForPAGE-SDS,resuspendsamplesinaminimalvolumeofsamplebuffer.(ThepresenceofsomeTCAcangiveayellowcolourasaconsequenceoftheacidificationofthesamplebuffer;titratewith1NNaOHor1MTrisHClpH8.5toobtainthenormalbluesamplebuffercolour.)NormalTCAToeliminateTCAsolubleinterferencesandproteinconcentration1)ToasampleofproteinsolutionaddTrichloroaceticacid(TCA)100%toget13%finalconcentration.Mixandkeep5min–20oCandthen15min4oC;orlongertimeat4oCwithoutthe–20oCstepforlowerproteinconcentration.Suggestion:leaveONiftheproteinconcentrationisverylow.(preparationof100%TCA:454mlH2O/kgTCA.Maintainindarkbottleat4oC.Becareful,usegloves!!!).2)Spin15min4oCinmicrofugeatmaximumspeed(15000g).Carefullydischargesupernatantandretainthepellet:drytubebyinversionontissuepaper(pelletmaybedifficulttosee).3)ForPAGE-SDS,resuspendsamplesinaminimalvolumeofsamplebuffer.(ThepresenceofsomeTCAcangiveayellowcolourasaconsequenceoftheacidificationofthesamplebuffer;titratewith1NNaOHor1MTrisHClpH8.5toobtainthenormalbluesamplebuffercolour.)AcetonePrecipitationToeliminateacetonesolubleinterferencesandproteinconcentration1)Addto1volumeofproteinsolution4volumesofcoldacetone.Mixandkeepatleast20min–20oC.(Suggestion:leaveONiftheproteinconcentrationisverylow).2)Spin15min4oCinmicrofugeatmaximumspeed(15000g).Carefullydischargesupernatantandretainthepellet:drytubebyinversionontissuepaper(pelletmaybedifficulttosee).3)Drysamplesundervaccum(speed-vac)ordryairtoeliminateanyacetoneresidue(smelltubes).ForPAGE-SDS,resuspendsamplesinaminimalvolumeofsamplebuffer.EthanolPrecipitationUsefulmethodtoconcentrateproteinsandremovalofGuanidineHydrochloridebeforePAGE-SDS1)Addto1volumeofproteinsolution9volumesofcoldEthanol100%.Mixandkeepatleast10min.at–20oC.(Suggestion:leaveON).2)Spin15min4oCinmicrocentrifugeatmaximumspeed(15000g).Carefullydischargesupernatantandretainthepellet:drytubebyinversionontissuepaper(pelletmaybedifficulttosee).3)Washpelletwith90%coldethanol(keepat–20oC).Vortexandrepelletsamples5minatfullspeed.4)Drysamplesundervaccum(speedvac)ordryairtoeliminateanyethanolresidue(smelltubes).ForPAGE-SDS,resuspendsamplesinaminimalvolumeofsamplebuffer.TCA-DOC/AcetoneUsefulmethodtoconcentrateproteinsandremoveacetoneandTCAsolubleinterferences1.Toonevolumeofproteinsolutionadd2%Nadeoxycholate(DOC)to0.02%final(for100μlsample,add1μl2%DOC).2.Mixandkeepatroomtemperatureforatleast15min.3.100%trichloroaceticacid(TCA)toget10%finalconcentration(preparationof100%TCA:454mlH2O/kgTCA.Maintainindarkbottleat4oC.Becareful,usegloves!!!).4.Mixandkeepatroomtemperatureforatleast1hour.5.Spinat4oCfor10min,removesupernatantandretainthepellet.Drytubebyinversionontissuepaper.6.Add200μloficecoldacetonetoTCApellet.7.Mixandkeeponiceforatleast15min.8.Spinat4oCfor10mininmicrocentrifugeatmaximumspeed.9.Removesupernatantasbefore(5),dryairpellettoeliminateanyacetoneresidue(smelltubes).ForPAGE-SDS,resuspendsamplesinaminimalvolumeofsamplebuffer.10.(ThepresenceofsomeTCAcangiveayellowcolourasaconsequenceoftheacidificationofthesamplebuffer;titratewith1NNaOHor1MTrisHClpH8.5toobtainthenormalbluesamplebuffercolour.)AcidifiedAcetone/MethanolUsefulmethodtoremoveacetoneandmethanolsolubleinterferenceslikeSDSbeforeIEF1)Prepareacidifiedacetone:120mlacetone+10μlHCl(1mMfinalconcentration).2)Prepareprecipitationreagent:Mixequalvolumesofacidifiedacetoneandmethanolandkeepat-20oC.3)Toonevolumeofproteinsolutionadd4volumesofcoldprecipitationreagent.MixandkeepONat-20oC.4)Spin15min4oCinmicrofugeatmaximumspeed(15000g).Carefullydischargesupernatantandretainthepellet:drytubebyinversionontissuepaper(pelletmaybedifficulttosee).5)Drysamplesundervaccum(speed-vac)ordryairtoeliminateanyacetoneormethanolresidue(smelltubes).TCA-EthanolPrecipitationUsefulmethodtoconcentrateproteinsandremovalofGuanidineHydrochloridebeforePAGE-SDS1)Dilute10-25μlsamplesto100μlwithH2OAdd100μlof20%trichloroaceticacid(TCA)andmix(preparationof100%TCA:454mlH2O/kgTCA.Maintainindarkbottleat4oC.Becareful,usegloves!!!).2)Leaveinicefor20min.Spinat4oCfor15mininmicrocentrifugeatmaximumspeed.3)Carefullydischargesupernatantandretainthepellet:drytubebyinversionontissue(pelletmaybedifficulttosee).4)Washpelletwith100μlice-coldethanol,dryandresuspendinsamplebuffer.5)IncasetherearetracesofGuHClpresent,samplesshouldbeloadedimmediatelyafterboilingfor7minat95°C6)(ThepresenceofsomeTCAcangiveayellowcolourasaconsequenceoftheacidificationofthesamplebuffer;tit