免疫共沉淀ChIP

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1、ChIPprotocolBasiconEZ-ChIP™ChromatinImmunoprecipitationKitUpstateCatalog#17-371A.InVivoCrosslinkingandLysisl1、Stimulateortreat4.5x107cellsona10cmdishasappropriate.(25K、5Kfor4h)l2、CrosslinkproteintoDNAbyaddingformaldehydedirectlytoculturemediumtoafinalconcentrationof1%andincubate10minutesatroom

2、temperature.(add270ul37%formaldehydeinto10mlofgrowthmediumonplate)l3.Add1mlof10Xglycinetoeachdishtoquenchunreactedformaldehyde.Swirltomixandincubateatroomtemperaturefor5minutes.l4、Aspiratemedium,washcellstwiceusing10mlicecoldPBS.l5、Add1mlcoldPBScontainingProteaseInhibitortodish.l6、Scrapecellsi

3、ntoconicaltube.Spinat700rcfat4℃for2-5minutestopelletcells.l7、Removesupernatant.l8、Resuspendeachcellpelletin600ulofSDSLysisBuffer(1%SDS,10mMEDTA,50mMTris,pH8.1)containingProteaseInhibitor.Incubateonicefor10minutes.(Remove5ulofcelllysateforunshearedDNAcontrol).(Lysatecanbefrozenat-80℃atthisstep.

4、)B.SonicationtoShearDNAl1、Sonicatechromatintoanaveragelengthofabout500bpwhilekeepingsamplesonice.(60%output,10s/6timesrecall#20program)l2、Microfugeat13,000rpmfor10minutesat4℃.Carefullyremovethesupernatantandtransfertoanewtubein100ulaliquots.(Shearedcrosslinkedchromatincanbestoredat-80°Cforupto

5、2months.)l3、Toallthesamples(unshearedandsheared),remove5ultoafreshtube.AddChIPelutionbuffer(1%SDS,0.1MNaHCO3)toafinalvolumeof30ul.l4、Add1ulofRNaseA(10mg/ml)andincubatefor30minutesat37℃.l5、Add1ulProteinaseK(10mg/ml)andincubateat62℃for2hour.l6、Load15ulona2%agarosegel.Observewhichoftheshearingcon

6、ditionsgivesasmearofDNAintherangeof200bp-1000bp.C.Immunoprecipitation(IP)ofCrosslinkedProtein/DNAl1、Add900ulofDilutionBuffer(0.01%SDS,1.1%TritonX-100,1.2mMEDTA,16.7mMTris-HCl,pH8.1,167mMNaCl,containingProteaseInhibitor)intoeachtubecontaining100ulofchromatin.l2、Preclear:add30ulofProteinG/AAgaro

7、seforeachIPincubate1hat4℃withrotation.(Afterpreclearremove50ulsupernatantforinputDNA)Pre-incubate:add30ulofProteinG/AAgarosewith2ugimmunoprecipitatingantibodyincubate1hat4℃withrotation.(c-junH79,ATF2SC187x).l3、Supernatantofprecleartubei