ChIPresultsobtainedwiththeantibodydirectedagainstH3K9me2
ChIPassayswereperformedusinghumanHeLacells,theantibodyagainstH3K9me2(Cat.#25270)andoptimizedPCRprimersetsforqPCR.ChIPwasperformedusingshearedchromatinfrom1millioncells.Atitrationoftheantibodyconsistingof1,2,5,and10µgperChIPexperimentwasanalyzed.IgG(2µg/IP)wasusedasnegativeIPcontrol.QPCRwasperformedwithprimersspecificforpromoteroftheinactiveHBBgeneandthecodingregionsoftheinactiveMYODgene,usedaspositivecontrols,andforthepromotersoftheactivegenesc-fosandGAPDH,usedasnegativecontrols.Figure1showstherecovery,expressedasa%ofinput(therelativeamountofimmunoprecipitatedDNAcomparedtoinputDNAafterqPCRanalysis).

Determinationoftheantibodytiter
Todeterminethetiteroftheantibody,anELISAwasperformedusingaserialdilutionoftheantibodyagainstH3K9me2(Cat.#25270),crudeserumandflow-through.Theantigenusedwasapeptidecontainingthehistonemodificationofinterest.Byplottingtheabsorbanceagainsttheantibodydilution(Figure2),thetiteroftheantibodywasestimatedtobe1:103,000.

CrossreactivitytestsusingtheantibodydirectedagainstH3K9me2
ADotBlotanalysiswasperformedtotestthecrossreactivityoftheantibodyagainstH3K9me2(Cat.#25270)withpeptidescontainingothermodificationsofhistoneH3andtheunmodifiedH3K9sequence.Onehundredto0.2pmolofpeptidecontainingtherespectivehistonemodificationwerespottedonamembrane.Theantibodywasusedatadilutionof1:20,000.Figure3showsahighspecificityoftheantibodyforthemodificationofinterest.

WesternblotanalysisusingtheantibodydirectedagainstH3K9me2
Histoneextracts(15µg)fromHeLacellswereanalyzedbyWesternblotusingtheantibodyagainstH3K9me2(Cat.#25270)diluted1:1,000inTBS-Tweencontaining5%skimmedmilk.Thepositionoftheproteinofinterestisindicatedontheright;theMarker(inkDa)isshownontheleft.

ImmunofluorescenceusingtheantibodydirectedagainstH3K9me2
MouseNIH3T3cellswerestainedwiththeantibodyagainstH3K9me2(Cat.#25270)andwithDAPI.Cellswerefixedwith4%formaldehydefor10min.andblockedwithPBS/TX-100containing5%normalgoatserumand1%BSA.ThecellswereimmunofluorescentlylabeledwiththeH3K9me2antibody(left)diluted1:500inblockingsolutionfollowedbyananti-rabbitantibodyconjugatedtoAlexa488.ThemiddlepanelshowsstainingofthenucleiwithDAPI.Amergeofthetwostainingsisshownontheright.

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