ChIPresultsobtainedwiththeantibodydirectedagainstH3K64me3
ChIPassayswereperformedusinghumanK562cells,theantibodyagainstH3K64me3(Cat.No.25260)andoptimizedPCRprimersetsforqPCR.ChIPwasperformedonshearedchromatinfrom1millioncells.Atitrationoftheantibodyconsistingof1,2,5,and10μgperChIPexperimentwasanalysed.IgG(2μg/IP)wasusedasnegativeIPcontrol.QPCRwasperformedwithprimersforthepromoteroftheactiveGAPDHandEIF4A2genes,usedasnegativecontrols,andfortheSat2andSatasatelliterepeats,usedaspositivecontrols.Figure1showstherecovery,expressedasa%ofinput(therelativeamountofimmunoprecipitatedDNAcomparedtoinputDNAafterqPCRanalysis).

Determinationoftheantibodytiter
Todeterminethetiter,anELISAwasperformedusingaserialdilutionoftheantibodydirectedagainstH3K64me3(Cat.No.25260)inantigencoatedwells.Theantigenusedwasapeptidecontainingthehistonemodificationofinterest.Byplottingtheabsorbanceagainsttheantibodydilution(Figure2),thetiteroftheantibodywasestimatedtobe1:5,500.

CrossreactivitytestsusingtheantibodydirectedagainstH3K64me3
TocheckthespecificityoftheantibodyagainstH3K64me3(Cat.No.25260)aDotBlotwasperformedwithpeptidescontainingdifferentmodificationsofhistoneH3andH4ortheunmodifiedH3K64sequence.Onehundredto0.2pmolofpeptidecontainingtherespectivehistonemodificationwerespottedonamembrane.Theantibodywasusedatadilutionof1:5,000.Figure3showsahighspecificityoftheantibodyforthemodificationofinterest.

WesternblotanalysisusingtheantibodydirectedagainstH3K64me3
Westernblotwasperformedonhistoneextracts(30μg)fromHeLacellsusingtheantibodyagainstH3K64me3(Cat.No.25260).Theantibodywasdiluted1:100inTBS-Tweencontaining5%skimmedmilk.TheMarker(inkDa)isshownontheleft,thepositionoftheproteinisindicatedontheright.

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