Chromatinimmunoprecipitation:atoolforstudyinghistoneacetylationandtranscriptionfactorbindingVirginiaA.Spencer,Jian-MinSun,LinLi,andJamesR.Davie*ManitobaInstituteofCellBiology,UniversityofManitoba,Winnipeg,Manitoba,CanadaR3E0V9Accepted19February2003AbstractThefunctionofaproteiningeneexpressioncanoftenbeexplained,inpart,bythelocationofthatproteinalongaspecificgenesequence.Inrecentyears,thechromatinimmunoprecipitation(ChIP)assayhasbeendevelopedtostudytheassociationofproteinslocatedwithin2�AofDNAsuchastranscriptionfactorsandmodifiedhistones.NumerousimportantfindingshavebeenpublishedusingtheChIPassayandmanyquestionsabouttranscriptionhavebeenanswered.Inthisarticle,wepresenttheChIPassaycurrentlyusedinourlabanddiscussthevariouswaystooptimizethisassayforone�sownuse.�2003PublishedbyElsevierScience(USA).Keywords:Chromatinimmunoprecipitation;Histone;Transcriptionfactor;Formaldehyde;Cisplatin1.IntroductionThechromatinimmunoprecipitation(ChIP)assayhasbecomeaverypopulartechniqueforfine-mappingthelocationofmodifiedhistones,transcriptionfactors,andnonhistonechromosomalproteins.Moreover,itisaninsitutechniquethatoffersamorephysiologicalrepresentationofnucleareventsinvolvedinthepro-cessingofDNA.Inthisassay,cellsareincubatedbrieflywiththecrosslinkerformaldehydetopreventthesepa-rationofDNA-associatedproteinsfromtheirtargetDNAsequenceinsubsequentsteps(Fig.1).ThecellsarethensonicatedtofragmenttheDNA,andthelysateiscentrifugedtoremoveinsolublecellulardebris.Theclearedlysateisincubatedwithaprimaryantibody,andtheantibody–antigencomplexesarethencapturedwithproteinA–orproteinG–Sepharosedependingonthenatureoftheantibody.TheSepharoseiswashedseveraltimeswithbufferscontainingdifferentsaltanddetergentconcentrations,andtheantibody–antigencomplexesareelutedfromtheproteinA/Gwithahighdetergentelu-tionbuffer.TheproteincomplementoftheimmunecomplexesisthendigestedawayandtheDNAisolatedbyethanolprecipitation.Initially,theChIPassaywasusedtostudytheasso-ciationofhyperacetylatedhistoneswithspecificDNAs...
