ChIPresultsobtainedwiththeantibodydirectedagainstH2AK119ub
ChIPassayswereperformedusinghumanHeLacells,theantibodyagainstH2AK119ub(cat.No.25229)andoptimizedPCRprimerpairsforqPCR.ChIPwasperformedusingshearedchromatinfrom1millioncellsontheSX-8GIP-Starautomatedsystem.Atitrationconsistingof1,2,5and10μgofantibodyperChIPexperimentwasanalyzed.IgG(2μg/IP)wasusedasanegativeIPcontrol.QuantitativePCRwasperformedwithprimersforthepromotersoftheactiveGAPDHandc-fosgenes,usedasnegativecontrols,andfortheinactiveMYT1andTSH2Bgenes,usedasapositivecontrols.Figure1showstherecovery,expressedasa%ofinput(therelativeamountofimmunoprecipitatedDNAcomparedtoinputDNAafterqPCRanalysis).

Determinationoftheantibodytiter
Todeterminethetiteroftheantibody,anELISAwasperformedusingaserialdilutionoftheantibodyagainstH2AK119ub(cat.No.25229).Theantigenusedwasapeptidecontainingthehistonemodificationofinterest.Byplottingtheabsorbanceagainsttheantibodydilution(Figure2),thetiteroftheantibodywasestimatedtobe1:23,000.

CrossreactivitytestsusingtheantibodydirectedagainstH2AK119ub
TotestthecrossreactivityoftheantibodyagainstH2AK119ub(cat.No.25229),aDotBlotanalysiswasperformedwithpeptidescontainingotherhistoneubiquitinylationsandunmodifiedsequences.Onehundredto0.2pmoloftherespectivepeptideswerespottedonamembrane.Theantibodywasusedatadilutionof1:20,000.Figure3showsahighspecificityoftheantibodyforthemodificationofinterest.

WesternblotanalysisusingtheantibodydirectedagainstH2AK119ub
WesternblotwasperformedonwholecellextractsfromHeLacells(25μg,lane1),andon1μgofrecombinanthistoneH2A,H2B,H3andH4(lane2,3,4and5,respectively)usingtheantibodyagainstH2AK119ub(cat.No.25229).Theantibodywasdiluted1:200inTBS-Tweencontaining5%skimmedmilk.TheMarker(inkDa)isshownontheleft.

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