科研相关软件chip

TargetGeneIdentification(individualandBindingSiteConsensusRNA--DNA--ProteinAnalysisofEpigeneticPhenomenon(eg,methylation, 不足以充分反映生理条件下

ChIP Treatcellswithformaldehyde:protein-proteinandprotein-DNAcross-linksstoptranscriptionfactorsintheirtracks(DAY0)Cross-linkingcanbedoneSuspensionAdherentAnatomicalSeeprotocolandwebpageforadditionalinformationcross-linkingandexampleDayDay1—Crosslinking，NucleipreparationandDigestionofchromatinSwellingBufferisusedtobloatcelltoallowcellmembranetobreakmoreeasilyduringDouncingDouncecontainstwoAislargeBissmalldispersescell“pokes”holesinSonicationSonication6112131Sonicationresultsin6112131103500100FragmentsizelimitsforEnzyme-basedEnzyme-basedDigestionvs.-basedImmunoprecipitationImmunoprecipitation(DAY1&AntibodiesareYshapedmoleculeswithbindingsitesforspecificantigens.Theheavychaincancomeinseveraldifferentclasses.HundredsofAbstoarecommerciallyChIPChIPAntibodyStep1:ShowreactivityonaWestern(denaturedStep2:ShowIPability.PerformIP,runWesternand-probewithsameAb(IP-Step3:Roughquantification,truebandshouldbe>50%ofcombinedsignalfromotherbands(ENCODEguidelines)PositivePositiveandNegativeIgGIgGpoolfromserum(DAYAnti-HistoneH3;rabbitmAb(DAY1)WashingWashing(DAYElutionElutionandCrosslinkAdditionofNaHCO3causesantibodiestoreleasetranscriptionfactors(DAY2)0.2MNaClat67ºfor3hoursormorereversescrosslinks,DNAfragmentsnowfreeinsolution(DAY2)RNaseAisaddeddigestRNAinsample(DAY3)PCRPurificationkitisusedtopurifyDNAforPCR,comparingpositiveAb,negativeAb,andInput(DAY3)PCRPCRtoVerifyChIP(DAYVerifyChIPofinterest(PolIIforus)isgreaterfortargetpromotercomparedtoInputvs.negativecontrolcomparedtoInputPositiveAbChIP(polII)ismuchhigherthannegativeAbChIP(IgG)fortargetpromotercomparedtonegativecontrolRememberthatifyouaredoingChIPonChip,youwillbeusingthepositiveAbChIPsamplesandtheInputaslabeledtargets,NOTthenegativeChIP(IgG)EfficiencyEfficiencyofNoAb(orIgGsupernatant)10ulInput+90ulBuffer=10%

+IP’ed,SonicatedChromatin100ul(eg)100%Input10%Input/50ul=0.2%Input/ul1

1ul

QIAquickpurifyElutein50ulEB(positive1ul

SonicatedChromatin100ul(eg)100%InputPositiveAb1 PercentInput=2%x2(C[T]2%InputSample–C[T]IPC[T]=CT=ThresholdcycleofPCRChIPSimpleChIP™SimpleChIP™ 货号：#8980SimpleChIPStemCellMasterRegulatorAssay用途：鉴定特定细胞类型中Oct-4，Sox2，Nanog与靶基因的结合情况。Oct-4ARabbitmAb#5677;Sox2XPRabbitmAb#5024;NanogXPRabbitmAb#5232;HumanOct-4PromoterPrimers#4641;HumanαSatelliteRepeatPrimers#4486储存：-20℃，干冰运输。保质期：2 货号：#8981SimpleChIPHumanBivalentPromoterAssay对(500l10Tri-Methyl-HistoneH3(Lys4)RabbitmAb#9751;Tri-Methyl-HistoneH3(Lys27)RabbitmAb#9733;Primers:TBD储存：-20℃，干冰运输。保质期：2SimpleChIP™AssayKits市场上独有，一个试剂盒中组合有2-3种抗体。而目前市场SimpleChIP™AssayKitsSimpleChIP™EnzymaticChromatinIPKit(AgaroseBeads)Czymai,T.etal.(2010)JBiolChem285,10163-78.Applications:ChIPDepartmentofDermatology,UniversityMedicalCenterMannheim,UniversityofHeidelberg,Theodor-Kutzer-Ufer1-3,68167Mannheim,Germany.Ghosh,R.etal.(2010)PLoSOne5,e9575.Applications:PrograminGeneFunctionandExpression,UniversityofMassachusettsMedicalSchool,Worcester,Massachusetts,UnitedStatesofAmerica.SimpleChIP™EnzymaticChromatinIPKit(MagneticBeads)Onishi,Y.(2010)BiosciRep,.Applications:InstituteforBiologicalResourcesandFunctions,NationalInstituteofAdvancedIndustrialScienceandTechnology(AIST),Tsukuba305-8566,Japan.y-DeBruyne,E.etal.(2010)Blood115,2430-40.Applications:DepartmentofHematologyandImmunology,VrijeUniversiteitBrussel,B-1090Brussels,Belgium.B