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1、PROTOCOLAnefficientchromatinimmunoprecipitation(ChIP)protocolforstudyinghistonemodificationsinArabidopsisplantsAbdelatySaleh1,2,Rau´lAlvarez-Venegas1,3&ZoyaAvramova11SchoolofBiologicalSciences,UniversityofNebraska,Lincoln,Nebraska68588,USA.2CenterforIntegratedFungalResearch(CIFR),DepartmentofPlan
2、tPathology,NorthCarolinaStateUniversity,Raleigh,NorthCarolina27695,USA.3DepartmentofGeneticEngineering,CentrodeInvestigacio´nydeEstudiosAvanzados,Campus-Guanajuato,Irapuato,C.P.36821,Me´xico.CorrespondenceshouldbeaddressedtoA.S.(asaleh@ncsu.edu)orZ.A.(zavramova2@unl.edu).sPublishedonline22May20
3、08;doi:10.1038/nprot.2008.66Chromatinimmunoprecipitation(ChIP)isapowerfultoolforthecharacterizationofcovalenthistonemodificationsandDNA–histoneinteractionsinvivo.TheprocedureincludesDNA–histonecross-linkinginchromatin,shearingDNAintosmallerfragments,immunoprecipitationwithantibodiesagainstthehis
4、tonemodificationsofinterest,followedbyPCRidentificationofassociatedDNAnatureprotocol/sequences.Inthisprotocol,wedescribeasimplifiedandoptimizedversionofChIPassaybyreducingthenumberofexperimentalmostepsandisolationsolutionsandshorteningpreparationtimes.Weincludeanuclearisolationstepbeforechromatins
5、hearing,whichc.erprovidesagoodyieldofhigh-qualityDNAresultinginatleast15lgofDNAfromeachimmunoprecipitatedsample(from0.2to0.4gutofstartingtissuematerial)sufficienttotestZ25genesofinterest.Thissimplerandcost-efficientprotocolhasbeenappliedforan.histone-modificationstudiesofvariousArabidopsisthaliana
6、tissuesandiseasytoadaptforothersystemsaswell.www//:ptINTRODUCTIONthCellsofamulticellularorganismaregeneticallysimilarbutstruc-proteins.However,thistechniqueislessreliableforDNAproteinpturallyandfunctionallydifferentbecauseofthedifferentialexpres-interactionsinheterologous(non-yeast)systemsanddi
7、splaysauorsionoftheirgenes.Overthepasttwodecades,increasingevidencetendencyforhighlevelsoffalse-positiveinteractions.GhassuggestedaroleforepigeneticmechanismsinthecontrolofTheChIPprocedureconsistsofdiscretestepsincludinginvivognid
