ChIPresultsobtainedwiththemonoclonalantibodydirectedagainstH3K4me2
ChIPassayswereperformedusingHeLacells,themonoclonalantibodyagainstH3K4me2(cat.No.25254)andoptimizedPCRprimersetsforqPCR.ChIPwasperformedusingshearedchromatinfrom1.6millioncells.Atitrationoftheantibodyconsistingof1,5and10μgperChIPexperimentwasanalysed.IgG(5μg/IP)wasusedasnegativeIPcontrol.QPCRwasperformedwithprimersforthepromoterandthecodingregionoftheGAPDHgene,andfortheRPL10andHBBpromoters.Figure1showstherecovery,expressedasa%ofinput(therelativeamountofimmunoprecipitatedDNAcomparedtoinputDNAafterqPCRanalysis).

CrossreactivityofthemonoclonalantibodydirectedagainstH3K4me2
TotestthespecificityanELISAwasperformedusingaserialdilutionofthemonoclonalantibodyagainstH3K4me2(cat.No.25254).ThewellswerecoatedwithpeptidescontainingtheunmodifiedH3K4aswellasthemono-,di-andtrimethylatedH3K4andthedimethylatedH3K9.Figure2showsahighspecificityoftheantibodyforthemodificationofinterest.

WesternblotanalysisusingthemonoclonalantibodydirectedagainstH3K4me2
Histoneextracts(15μg)fromHeLacellswereanalysedbyWesternblotusingthemonoclonalantibodyagainstH3K4me2(cat.No.25254)diluted1:1,000inTBS-Tweencontaining5%skimmedmilk.Thepositionoftheproteinofinterestisindicatedontheright;theMarker(inkDa)isshownontheleft.

ImmunofluorescenceusingthemonoclonalantibodydirectedagainstH3K4me2
HeLacellswerestainedwiththeantibodyagainstH3K4me2(cat.No.25254)andwithDAPI.Cellswerefixedwith4%formaldehydefor10minutesandblockedwithPBS/TX-100containing5%normalgoatserumand1%BSA.ThecellswereimmunofluorescentlylabelledwiththeH3K4me2antibody(left)diluted1:500inblockingsolutionfollowedbyananti-mouseantibodyconjugatedtoAlexa594.ThemiddlepanelshowsstainingofthenucleiwithDAPI.Amergeofthetwostainingsisshownontheright.

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