ChIPresultsobtainedwiththemonoclonalantibodydirectedagainstH3K27me3
ChIPassayswereperformedusinghumanHeLacells,themonoclonalantibodyagainstH3K27me3(Cat.No.25243)andoptimizedPCRprimersetsforqPCR.ChIPwasperformedonshearedchromatinfrom1millioncells.Atitrationoftheantibodyconsistingof1,2,5,and10μgperChIPexperimentwasanalysed.IgG(2μg/IP)wasusedasnegativeIPcontrol.QPCRwasperformedwithprimersforthepromotersoftheactivegenesc-fosandGAPDHasnegativecontrols,andforthecodingregionsoftheinactivegenesMYT1andTSH2Baspositivecontrols.Figure1showstherecovery,expressedasa%ofinput(therelativeamountofimmunoprecipitatedDNAcomparedtoinputDNAafterqPCRanalysis).TheseresultsareinaccordancewiththeobservationthatH3K27me3ispreferablypresentatinactivegenes.

CrossreactivityofthemonoclonalantibodydirectedagainstH3K27me3
TotestthespecificityanELISAwasperformedusingaserialdilutionofthemonoclonalantibodyagainstH3K27me3(Cat.No.25243).ThewellswerecoatedwithpeptidescontainingtheunmodifiedH3K27regionaswellasthemono-,di-andtrimethylatedH3K27andthetrimethylatedH3K9.Figure2showsahighspecificityoftheantibodyforthepeptidecontainingthemodificationofinterest.

WesternblotanalysisusingthemonoclonalantibodydirectedagainstH3K27me3
Histoneextracts(15μg)fromHeLacellswereanalysedbyWesternblotusingthemonoclonalantibodyagainstH3K27me3(Cat.No.25243)diluted1:1,000inTBS-Tweencontaining5%skimmedmilk.Thepositionoftheproteinofinterestisindicatedontheright;theMarker(inkDa)isshownontheleft.

ImmunofluorescenceusingthemonoclonalantibodydirectedagainstH3K27me3
HeLacellswerestainedwiththeantibodyagainstH3K27me3(Cat.No.25243)andwithDAPI.Cellswerefixedwith4%formaldehydefor10minutesandblockedwithPBS/TX-100containing5%normalgoatserumand1%BSA.ThecellswereimmunofluorescentlylabelledwiththeH3K27me3antibody(left)diluted1:500inblockingsolutionfollowedbyananti-mouseantibodyconjugatedtoAlexa594.ThemiddlepanelshowsstainingofthenucleiwithDAPI.Amergeofthetwostainingsisshownontheright.

Storeat–80°Cforupto2years.Centrifugeafterfirstthawtomaximizeproductrecovery.Aliquottoavoidrepeatedfreeze/thawcycles.Aliquotsmaybestoredat–20°Cforatleastonemonth.