ChIPresultsobtainedwiththeantibodydirectedagainstH3K9me3S10p
ChIPassayswereperformedusinghumanHeLacellstreatedwithcolcemid,theantibodyagainstH3K9me3S10p(Cat.#25273)andoptimizedPCRprimersetsforqPCR.ChIPwasperformedusingshearedchromatinfrom10,000cellsperIP.Atitrationoftheantibodyconsistingof1,5,and10µlperChIPexperimentwasanalyzed.Additionally,thesametitrationwasanalyzedafterincubationoftheantibodywith5nmolofthecorrespondingblockingpeptidefor1houratroomtemperature.IgG(5µg/IP)wasusedasnegativeIPcontrol.QPCRwasperformedwithprimersforthepromoteroftheactivegenesGAPDHandc-fosandfortheheterochromatinMarkerSat2.Figure1showstherecovery,expressedasa%ofinput(therelativeamountofimmunoprecipitatedDNAcomparedtoinputDNAafterqPCRanalysis).

Determinationofthetiter
Todeterminethetiter,anELISAwasperformedusingaserialdilutionoftheantibodydirectedagainsthumanH3K9me3S10p(Cat.#25273).Theantigenusedwasapeptidecontainingthehistonemodificationofinterest.Byplottingtheabsorbanceagainsttheantibodydilution(Figure2),thetiteroftheantibodywasestimatedtobe1:87,000.

CrossreactivitytestusingtheantibodydirectedagainstH3K9me3S10p
ADotBlotanalysiswasperformedtotestthecrossreactivityoftheantibodyagainstH3K9me3S10p(Cat.#25273)withpeptidescontainingothermodificationsandunmodifiedsequencesofhistoneH3.Onehundredto0.2pmolofthepeptidecontainingtherespectivehistonemodificationwerespottedonamembrane.Theantibodywasusedatadilutionof1:1,000.Figure3showsahighspecificityoftheantibodyforthemodificationofinterest.

WesternblotanalysisusingtheantibodydirectedagainstH3K9me3S10p
HeLacellsweretreatedwithcolcemidtoblockthecellcycleinmetaphaseand15µgofhistoneextractsofthesecellswereanalyzedbyWesternblotwiththeantibodyagainstH3K9me3S10p(Cat.#25273)diluted1:500inTBS-Tweencontaining5%skimmedmilk.Thepositionoftheproteinofinterestisindicatedontheright;themarker(inkDa)isshownontheleft.TheresultoftheWesternanalysiswiththeantibodyisshowninlane2;lane1showsthesameanalysisafterincubationoftheantibodywith5nmolofthecorrespondingblockingpeptidefor1houratroomtemperature.

ImmunofluorescenceusingtheantibodydirectedagainstH3K9me3S10p
HeLacellswerestainedwiththeantibodyagainstH3K9me3S10p(Cat.#25273)andwithDAPI.Cellswerefixedwith4%formaldehydefor10min.andblockedwithPBS/TX-100containing5%normalgoatserumand1%BSA.ThecellswereimmunofluorescentlylabelledwiththeH3K9me3S10pantibody(left)diluted1:500inblockingsolutionfollowedbyananti-rabbitantibodyconjugatedtoAlexa488.ThemiddlepanelshowsstainingofthenucleiwithDAPI.Amergeofthetwostainingsisshownontheright.

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