Determinationoftheantibodytiter
Todeterminethetiteroftheantibody,anELISAwasperformedusingaserialdilutionoftheantibodydirectedagainstH2A.XS139p(Cat.No.25227)inantigencoatedwells.Theantigenusedwasapeptidecontainingthehistonemodificationofinterest.Byplottingtheabsorbanceagainsttheantibodydilution(Figure1),thetiteroftheantibodywasestimatedtobe1:170,000.

CrossreactivitytestsusingtheantibodydirectedagainstH2A.XS139p
TotestthecrossreactivityoftheantibodyagainstH2A.XS139p(Cat.No.25227),aDotBlotanalysiswasperformedwithpeptidescontainingotherhistonephosphorylationsandtheunmodifiedH2A.X.Onehundredto0.2pmoloftherespectivepeptideswerespottedonamembrane.Theantibodywasusedatadilutionof1:10,000.Figure2showsahighspecificityoftheantibodyforthemodificationofinterest.

WesternblotanalysisusingtheantibodydirectedagainstH2A.XS139p
Westernblotwasperformedonhistoneextracts(15μg)fromuntreatedU2OScells(lane1)orfromU2OScellstreatedwithcamptothecin(lane2),andon1μgofrecombinanthistoneH2A,H2B,H3andH4(lane3,4,5and6,respectively)usingtheantibodyagainstH2A.XS139p(Cat.No.25227).Theantibodywasdiluted1:1,000inTBS-Tweencontaining5%skimmedmilk.TheMarker(inkDa)isshownontheleft.

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