ChIPresultsobtainedwiththemonoclonalantibodydirectedagainstH3K9me2
ChIPassayswereperformedusingHeLacells,themonoclonalantibodyagainstH3K9me3(Cat.No.25269)andoptimizedPCRprimersetsforqPCR.ChIPwasperformedusingshearedchromatinfrom1.6millioncells.Atitrationoftheantibodyconsistingof1,3and9μgperChIPexperimentwasanalysed.IgG(5μg/IP)wasusedasnegativeIPcontrol.QPCRwasperformedwithprimersforthepromoterandthecodingregionoftheGAPDHgene,andfortheRPL10andHBBpromoters.Figure1showstherecovery,expressedasa%ofinput(therelativeamountofimmunoprecipitatedDNAcomparedtoinputDNAafterqPCRanalysis).

CrossreactivityofthemonoclonalantibodydirectedagainstH3K9me2
TotestthespecificityanELISAwasperformedusingaserialdilutionofthemonoclonalantibodyagainstH3K9me2(Cat.No.25269).ThewellswerecoatedwithpeptidescontainingtheunmodifiedH3K9aswellasthemono-,di-andtrimethylatedH3K9andthedimethylatedH3K4andH3K27.Figure2showsahighspecificityoftheantibodyforthemodificationofinterest.

WesternblotanalysisusingthemonoclonalantibodydirectedagainstH3K9me2
Histoneextracts(15μg)fromHeLacellswereanalysedbyWesternblotusingthemonoclonalantibodyagainstH3K9me2(Cat.No.25269)diluted1:1,000inTBS-Tweencontaining5%skimmedmilk.Thepositionoftheproteinofinterestisindicatedontheright;theMarker(inkDa)isshownontheleft.

ImmunofluorescenceusingthemonoclonalantibodydirectedagainstH3K9me2
HeLacellswerestainedwiththeantibodyagainstH3K9me2(Cat.No.25269)andwithDAPI.Cellswerefixedwith4%formaldehydefor10minutesandblockedwithPBS/TX-100containing5%normalgoatserumand1%BSA.ThecellswereimmunofluorescentlylabelledwiththeH3K9me2antibody(left)diluted1:500inblockingsolutionfollowedbyananti-mouseantibodyconjugatedtoAlexa594.ThemiddlepanelshowsstainingofthenucleiwithDAPI.Amergeofthetwostainingsisshownontheright.

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