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1、PROTOCOLProtocolforthefastchromatinimmunoprecipitation(ChIP)methodJoelDNelson1,2,OlegDenisenko2&KarolBomsztyk1,21MolecularandCellularBiologyProgramand2UWMedicineatLakeUnion,UniversityofWashington,Seattle,Washington98109,USA.CorrespondenceshouldbeaddressedtoK.
2、B.(karolb@u.washington.edu).Publishedonline27June2006;doi:10.1038/nprot.2006.27Chromatinandtranscriptionalprocessesareamongthemostintensivelystudiedfieldsofbiologytoday.Theintroductionofchromatinimmunoprecipitations(ChIP)representsamajoradvancementinthisarea.T
3、hispowerfulmethodallowsresearcherstoprobespecificprotein-DNAinteractionsinvivoandtoestimatethedensityofproteinsatspecificsitesgenome-wide.WehaveintroducedseveralotocolsimprovementstothetraditionalChIPassay,whichsimplifytheprocedure,greatlyreducingthetimeandlabo
4、rrequiredtocompletertheassay.Thesimplicityofthemethodyieldshighlyreproducibleresults.Ourimprovementsfacilitatetheprobingofmultipleeprproteinsinasingleexperiment,whichallowsforthesimultaneousmonitoringofmanygenomicevents.Thismethodisparticularlyusefulinkinetic
5、studieswheremultiplesamplesareprocessedatthesametime.Startingwithshearedchromatin,PCR-readyDNAcanbeisolatedfrom16–24ChIPsamplesin4–6husingthefastmethod.e.com/naturINTRODUCTION.natuChromatiniscomposedofDNA,proteinsandRNA13.ChromatinproteinaseKdigestion,aproced
6、uredoneinonetube(Fig.1wstructureisdynamic,respondstointra-andextracellularsignals,andref.9).andcontrolsgeneexpression,DNAreplicationandrepair35.ChIPInaddition,weintroducetheuseofanultrasonicbathtohasproventobeapowerfulassaybywhichtofollowtheprotein-increaseth
7、erateofprotein-antibodybinding23sothatfortheDNAinteractionsinvolvedintheseprocesses3,68.Theassaydeter-antibodiesthatwetested,incubationtimewasdecreasedfrommineswhetheraprotein-DNAinteractionispresentatagivenZ1hdownto15min(refs.9,10andFig.2).ouphttp://wwrlocat
8、ionwithinthegenomeandallowsestimationofthedensityofOneoftheadvantagesofthefastChIPmethodisthattheagivenfactorinthatregion9,10.Alongwithothertechniques11,12,reducedamountoflaborpertubeallo
