ChIPresultsobtainedwiththeantibodydirectedagainstH3K9me1
ChIPassayswereperformedusinghumanHeLacells,theantibodydirectedagainstH3K9me1(Cat.No.25268)andoptimizedPCRprimersetsforqPCR.ChIPwasperformedusingshearedchromatinfrom10,000cells.Respectively1and5μgoftheantibodyand5μgofIgG(negativeIPcontrol)wereusedperChIPexperiment.QPCRwasperformedwithprimersfortheGAPDHpromoterandfortheinactivegeneMYOD.Figure1showstherecovery,expressedasa%ofinput(therelativeamountofimmunoprecipitatedDNAcomparedtoinputDNAafterqPCRanalysis).TheseresultsareinaccordancewiththeobservationthatH3K9me1ispreferablypresentatsilentgenes.

Determinationoftheantibodytiter
Todeterminethetiteroftheantibody,anELISAwasperformedusingaserialdilutionoftheantibodydirectedagainsthumanH3K9me1(Cat.No.25268),crudeserumandflowthroughinantigencoatedwells.Theantigenusedwasapeptidecontainingthehistonemodificationofinterest.Byplottingtheabsorbanceagainsttheantibodydilution(Figure2),thetiteroftheantibodywasestimatedtobe1:68,000.

CrossreactivitytestoftheantibodydirectedagainstH3K9me1
ADotBlotanalysiswasperformedtotestthecrossreactivityoftheantibodyagainstH3K9me1(Cat.No.25268)withpeptidescontainingothermodificationsandtheunmodifiedsequenceofhistoneH3.Onehundredto0.2pmolofthepeptidecontainingtherespectivehistonemodificationwerespottedonamembrane.Theantibodywasusedatadilutionof1:20,000.Figure3showsahighspecificityoftheantibodyforthemodificationofinterest.

WesternblotanalysisusingtheantibodydirectedagainstH3K9me1
Histoneextracts(15μg)fromHeLacellswereanalysedbyWesternblotusingtheantibodyagainstH3K9me1(Cat.No.25268)diluted1:1,000inTBS-Tweencontaining5%skimmedmilk.Thepositionoftheproteinofinterestisindicatedontheright;theMarker(inkDa)isshownontheleft.

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