ChIPresultsobtainedwiththeantibodydirectedagainstH3K9me3
ChIPassayswereperformedusinghumanHeLacells,theantibodyagainstH3K9me3(Cat.No.25272)andoptimizedPCRprimersetsforqPCR.ChIPwasperformedonshearedchromatinfrom1millionHeLaS3cells.Atitrationoftheantibodyconsistingof1,2,5,and10μgperChIPexperimentwasanalysed.IgG(2μg/IP)wasusedasnegativeIPcontrol.QPCRwasperformedwithprimersfortheheterochromatinMarkerSat2andfortheZNF510gene,usedaspositivecontrols,andforthepromotersoftheactivec-fosandGAPDHgenes,usedasnegativecontrols.Figure1showstherecovery,expressedasa%ofinput(therelativeamountofimmunoprecipitatedDNAcomparedtoinputDNAafterqPCRanalysis).

ChIP-seqresultsobtainedwiththeantibodydirectedagainstH3K9me3
ChIPwasperformedwith0.5μgoftheantibodyagainstH3K9me3(Cat.No.25272)onshearedchromatinfrom1millionHeLacells.TheimmunoprecipitatedDNAwasanalysedbyQPCRwithoptimizedPCRprimerpairsforthepromoterregionsoftheactiveGAPDHandEIF4A2genes,forthecodingregionoftheZNF510geneandfortheSat2satelliterepeat(figure2A).TheimmunoprecipitatedDNAwassubsequentlyanalysedonanIlluminaGenomeAnalyzer.Librarypreparation,clustergenerationandsequencingwereperformedaccordingtothemanufacturer’sinstructions.The36bptagswerealignedtothehumangenomeusingtheBWAalgorithm.Figure2Bshowsthesignaldistributionalongthelongarmofchromosome19andazoomintoanenrichedregioncontainingseveralZNFrepeatgenes.Figure2CandDshowtheenrichmentatZNF510andZNF12onchromosome9and7,respectively.TheseresultsclearlyshowanenrichmentofH3K9me3atZNFrepeatgenes.

Determinationoftheantibodytiter
Todeterminethetiteroftheantibody,anELISAwasperformedusingaserialdilutionoftheantibodydirectedagainsthumanH3K9me3(Cat.No.25272),crudeserumandflowthroughinantigencoatedwells.Theantigenusedwasapeptidecontainingthehistonemodificationofinterest.Byplottingtheabsorbanceagainsttheantibodydilution(Figure3),thetiteroftheantibodywasestimatedtobe1:60,000.

CrossreactivitytestsusingtheantibodydirectedagainstH3K9me3
ADotBlotanalysiswasperformedtotestthecrossreactivityoftheantibodyagainstH3K9me3(Cat.No.25272)withpeptidescontainingothermodificationsandunmodifiedsequencesofhistoneH3andH4.Onehundredto0.2pmolofthepeptidecontainingtherespectivehistonemodificationwerespottedonamembrane.Theantibodywasusedatadilutionof1:20,000.Figure4showsahighspecificityoftheantibodyforthemodificationofinterest.

WesternblotanalysisusingtheantibodydirectedagainstH3K9me3
WesternblotanalysiswasperformedonhistoneextractsfromHeLacells(15μg,lane1)andonrecombinantH3(1μg,lane2)usingtheantibodyagainstH3K9me3(Cat.No.25272).Theantibodywasdiluted1:1,000inTBS-Tweencontaining5%skimmedmilk.Thepositionoftheproteinofinterestisindicatedontheright;themarker(inkDa)isshownontheleft.

ImmunofluorescenceusingtheantibodydirectedagainstH3K9me3
HeLacellswerestainedwiththeantibodyagainstH3K9me3(Cat.No.25272)andwithDAPI.Cellswerefixedwith4%formaldehydefor10minutesandblockedwithPBS/TX-100containing5%normalgoatserumand1%BSA.ThecellswereimmunofluorescentlylabelledwiththeH3K9me3antibody(left)diluted1:500inblockingsolutionfollowedbyananti-rabbitantibodyconjugatedtoAlexa488.ThemiddlepanelshowsstainingofthenucleiwithDAPI.Amergeofthetwostainingsisshownontheright.

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