ChIPresultsobtainedwiththeantibodydirectedagainstH3K27me2
ChIPassayswereperformedusinghumanHeLacells,theantibodyagainstH3K27me2(Cat.#25241)andoptimizedPCRprimersetsforqPCR.ChIPwasperformedusingshearedchromatinfrom1millioncells.Atitrationoftheantibodyconsistingof1,2,5,and10µgperChIPexperimentwasanalyzed.IgG(2µg/IP)wasusedasnegativeIPcontrol.QPCRwasperformedwithprimersforthepromoteroftheactiveGAPDHandEIF4A2genes,usedasnegativecontrols,andforthepromoteroftheinactiveHBBandthecodingregionoftheinactiveMYOD1genes,usedaspositivecontrols.Figure1showstherecovery,expressedasa%ofinput(therelativeamountofimmunoprecipitatedDNAcomparedtoinputDNAafterqPCRanalysis).TheseresultsareinaccordancewiththeobservationthatH3K27me2ispreferablypresentatsilentgenes.

Determinationoftheantibodytiter
Todeterminethetiteroftheantibody,anELISAwasperformedusingaserialdilutionoftheantibodydirectedagainstH3K27me2(Cat.#25241).Theantigenusedwasapeptidecontainingthehistonemodificationofinterest.Byplottingtheabsorbanceagainsttheantibodydilution(Figure2),thetiteroftheantibodywasestimatedtobe1:480,000.

CrossreactivitytestsusingtheantibodydirectedagainstH3K27me2
ADotBlotanalysiswasperformedtotestthecrossreactivityoftheantibodyagainstH3K27me2(Cat.#25241)withpeptidescontainingothermodificationsofhistoneH3andH4andtheunmodifiedsequence.Onehundredto0.2pmolofpeptidecontainingtherespectivehistonemodificationwerespottedonamembrane.Theantibodywasusedatadilutionof1:50,000.Figure3showsahighspecificityoftheantibodyforthemodificationofinterest.

WesternblotanalysisusingtheantibodydirectedagainstH3K27me2
Histoneextracts(15µg)fromHeLacellswereanalyzedbyWesternblotusingtheantibodyagainstH3K27me2(Cat.#25241)diluted1:1,000inTBS-Tweencontaining5%skimmedmilk.Thepositionoftheproteinofinterestisindicatedontheright;theMarker(inkDa)isshownontheleft.

ImmunofluorescenceusingtheantibodydirectedagainstH3K27me2
HeLacellswerestainedwiththeantibodyagainstH3K27me2(Cat.#25241)andwithDAPI.Cellswerefixedwith4%formaldehydefor10min.andblockedwithPBS/TX-100containing5%normalgoatserumand1%BSA.ThecellswereimmunofluorescentlylabelledwiththeH3K27me2antibody(left)diluted1:500inblockingsolutionfollowedbyananti-rabbitantibodyconjugatedtoAlexa488.ThemiddlepanelshowsstainingofthenucleiwithDAPI.Amergeofthetwostainingsisshownontheright.

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