染色质免疫共沉淀 ChIP Protocol及crosslink 的原理.doc

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1、ChIPProtocolI.Formaldehydecross-linkingofcellsUse5x107–1x108cells(70-80%confluencyforadhesioncellsof8-1215cm2platesor175cm2flasks)foreachimmunoprecipitation.Adherentcells:1.Add1/10volumeoffresh11%FormaldehydeSolutiontoplates.2.Swirlplatesbrieflyandletthemsitatroomtemperaturefor10mi

2、nutes.3.Add1/20volumeof2.5Mglycinetoplatestoquenchformaldehyde.4.Rinsecellstwicewith5ml1xPBS.Harvestcellsusingsiliconscraper.5.Poolcellsin50mlconicaltubesandspinat1,350xgfor5minutesat4°C(SorvallLegendRTcentrifugewithswingingbucketrotor).Discardsupernatantandresuspendpelletin10ml1xP

3、BSper108cells.6.Transfer5x107–1x108cellsto15mlconicaltubeandspinat1,350xgfor5minutesat4°C(SorvallLegendRTcentrifugewithswingingbucketrotor).Discardsupernatant.7.Flashfreezecellsinliquidnitrogenandstorepelletsat–80°C.Suspensioncells:1.Add1/10volumeoffresh11%FormaldehydeSolutiontofla

4、sks.2.Swirlflasksbrieflyandletthemsitatroomtemperaturefor20minutes.3.Add1/20volumeof2.5Mglycinetoflaskstoquenchformaldehyde.4.Spincellsat1,350xgfor5minutesat4°C.5.Poolcellsinrequirednumberof50mlconicaltubesandspinat1,350xgfor5minutesat4°C(SorvallLegendRTcentrifugewithswingingbucket

5、rotor).Dumpsupernatant.6.Resuspendcellsin50ml1XPBS,spinat1,350xgfor5minutesat4°C.Discardsupernatant.Repeatonce.7.Resuspendin10mlper108cells.Aliquot5x107–1x108cellsto15mlconicaltubesandspin1,350xgfor5minutesat4°C(SorvallLegendRTcentrifugewithswingingbucketrotor).Discardsupernatant.8

6、.Flashfreezecellsinliquidnitrogenandstorepelletsat–80°C.FormaldehydeSolutionFinalConc.StockFor50ml50mM1MHepes-KOH,pH7.52.5ml100mM5MNaCl1.0ml1mM0.5MEDTA50.0µl0.5mM0.5MEGTA100.0µl11%37%Formaldehyde14.9mlddH2O31.5mlII.PreblockandbindingofantibodytomagneticbeadsNOTE:ExacttypeofDynalbea

7、d(ProteinA,ProteinG,sheepanti-mouseIgG,sheepanti-rabbitIgG,etc.)dependsonantibodybeingused.Youcantryotherbrands/kindsofbeads,butwehavenoinformationaboutwhatchangesyouwouldhavetomakeintheprotocolorwhatkindsofeffectsthesechangeswouldhaveontheresults.1.Add100µlDynalmagneticbeadstomicr

8、ofugetube.Add1mlblocksolut