TCA-DOCForprecipitationofverylowproteinconcentration

1)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.)

NormalTCAToeliminateTCAsolubleinterferencesandproteinconcentration

1)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.)

AcetonePrecipitationToeliminateacetonesolubleinterferencesandproteinconcentration

1)Addto1volumeofproteinsolution4volumesofcoldacetone.Mixandkeepatleast20min–20ºC.(Suggestion:leaveONiftheproteinconcentrationisverylow).2)Spin15min4ºCinmicrofugeatmaximumspeed(15000g).Carefullydischargesupernatantandretainthepellet:drytubebyinversionontissuepaper(pelletmaybedifficulttosee).3)Drysamplesundervaccum(speed-vac)ordryairtoeliminateanyacetoneresidue(smelltubes).ForPAGE-SDS,resuspendsamplesinaminimalvolumeofsamplebuffer.

EthanolPrecipitationUsefulmethodtoconcentrateproteinsandremovalofGuanidineHydrochloridebeforePAGE-SDS

1)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/AcetoneUsefulmethodtoconcentrateproteinsandremoveacetoneandTCAsolubleinterferences

1.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/MethanolUsefulmethodtoremoveacetoneandmethanolsolubleinterferenceslikeSDSbeforeIEF

1)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-SDS

1)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;titratewith1NNaOHor1MTrisHClpH8.5toobtainthenormalbluesamplebuffercolour.)PAGEprep^TMProteinClean-upandEnrichmentKit-Pierce

ThePAGEprep™KitenablesremovalofmanychemicalsthatinterferewithSDS-PAGEanalysis:guanidine,ammoniumsulfate,othercommonsalts,acidsandbases,detergents,dyes,DNA,RNA,andlipids.

PIERCE:#26800-PAGEprep^TMProteinClean-upandEnrichmentKit(pdf)

ChloroformMethanolPrecipitationUsefulmethodforRemovalofsaltanddetergents

1)Tosampleofstartingvolume100ul2)Add400ulmethanol3)Vortexwell4)Add100ulchloroform5)Vortex6)Add300ulH2O7)Vortex8)Spin1minute@14,0000g9)Removetopaqueouslayer(proteinisbetweenlayers)10)Add400ulmethanol11)Vortex12)Spin2minutes@14,000g13)RemoveasmuchMeOHaspossIBLewithoutdisturbingpellet14)Speed-Vactodryness15)Bringupin2XsamplebufferforPAGEReference:Wessel,D.andFlugge,U.I.Anal.Biochem.(1984)138,141-143