ChIPresultsobtainedwiththeantibodydirectedagainstH4K20me1
ChIPassayswereperformedusinghumanHeLacells,theantibodyagainstH4K20me1(cat.No.25280)andoptimizedPCRprimersetsforqPCR.ChIPwasperformedonshearedchromatinfrom1millioncells.Atitrationoftheantibodyconsistingof1,2,5,and10μgperChIPexperimentwasanalysed.IgG(2μg/IP)wasusedasnegativeIPcontrol.QPCRwasperformedwithprimersforthecodingregionoftheactiveGAPDHandACTBgenes,usedaspositivecontrols,andfortheGAPDHpromoterandaregionlocated1kbupstreamoftheGAPDHpromoter,usedasnegativecontrols.Figure1showstherecovery,expressedasa%ofinput(therelativeamountofimmunoprecipitatedDNAcomparedtoinputDNAafterqPCRanalysis).

Determinationoftheantibodytiter
Todeterminethetiter,anELISAwasperformedusingaserialdilutionoftheantibodydirectedagainstH4K20me1(cat.No.25280)inantigencoatedwells.Theantigenusedwasapeptidecontainingthehistonemodificationofinterest.Byplottingtheabsorbanceagainsttheantibodydilution(Figure2),thetiteroftheantibodywasestimatedtobe1:51,100.

CrossreactivitytestsusingtheantibodydirectedagainstH4K20me1
TocheckthespecificityoftheantibodyagainstH4K20me1(cat.No25280)aDotBlotwasperformedwithpeptidescontainingdifferentmodificationsofhistoneH3andH4ortheunmodifiedH4K20sequence.Onehundredto0.2pmolofpeptidecontainingtherespectivehistonemodificationwerespottedonamembrane.Theantibodywasusedatadilutionof1:20,000.Figure3showsahighspecificityoftheantibodyforthemodificationofinterest.

WesternblotanalysisusingtheantibodydirectedagainstH4K20me1
Westernblotwasperformedonwholecellextracts(25μg,lane1)fromHeLacells,andon1μgofrecombinanthistoneH2A,H2B,H3andH4(lane2,3,4and5,respectively)usingtheantibodyagainstH4K20me1(cat.No.25280).Theantibodywasdiluted1:1,000inTBS-Tweencontaining5%skimmedmilk.TheMarker(inkDa)isshownontheleft,thepositionoftheproteinisindicatedontheright.

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