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1、PublishedonlineJanuary5,2006NucleicAcidsResearch,2006,Vol.34,No.1e2doi:10.1093/nar/gnj004Fastchromatinimmunoprecipitationassay1,2221,2,JoelD.Nelson,OlegDenisenko,PavelSovaandKarolBomsztyk*12MolecularandCellularBiologyProgramandUWMedicineLakeUnionResearch,UniversityofWashington,
2、Seattle,WA98109,USADownloadedfromReceivedNovember15,2005;RevisedandAcceptedDecember12,2005ABSTRACTMATERIALSANDMETHODSChromatinimmunoprecipitation(ChIP)isawidelyMammaliancellshttp://nar.oxfordjournals.org/usedmethodtoexploreinvivointeractionsbetweenRatmesangialcellsweregrownin15
3、0mmplasticcellcultureproteinsandDNA.TheChIPassaytakesseveraldaysdishesinRPMI1640mediasupplementedwith10%FBS,tocomplete,involvesseveraltubetransfersanduses2mMglutamine,penicillin(100U/ml),streptomycin(0.01%)eitherphenol–chlorophormorspincolumnstopurifyandhumidifiedwith7/93%ofCO2/
4、airgasmixture(14).DNA.ThetraditionalChIPmethodbecomesachal-lengewhenhandlingmultiplesamples.WehaveYeaststrainsdevelopedanefficientandrapidChelexresin-basedMediausedforthegrowthofSaccharomycescerevisiaewereChIPprocedurethatdramaticallyreducestimeofthepreviouslydescribed(15);cell
5、sweregrownat30C.ThestrainatLibraryoftheThirdSchoolofClinicalMedicalofPekingUniversityonSeptember22,2012assayandusesonlyasingletubetoisolatePCR-readyusedinthisstudywasMATaHMLaHMRaade2-101his3-DNA.ThismethodgreatlyfacilitatestheprobingofD200leu2-D1lys2-801trp1D-1ura3-52(16).chro
6、matinchangesovermanytimepointswithseveralantibodiesinoneexperiment.ReagentsAntibodiesandblockingpeptides.TheantibodytotheC-terminalpeptideofKproteinwasraisedinrabbitsasdescribedINTRODUCTIONbefore(17).Anti-Sir2pserumwasagiftfromJasperRine,UniversityofCalifornia,Berkeley(18).Theo
7、therantibodiesChromatiniscomposedofDNA,proteinsandRNA(13).used,anti-RNApolymeraseII,anti-histoneH3andrabbitIgGChromatinstructureisdynamic,respondstoextracellularsig-werefromSantaCruz(cat#sc-899),Abcam(cat#1791),andnals,andcontrolsgeneexpression,celldivisionandDNAVectorLaborator
8、ies(cat#I-1000),respectively.Theblockingrepair(35).Chr
