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Determinationofthetiter
Todeterminethetiter,anELISAwasperformedusingaserialdilutionoftheantibodydirectedagainstH3K23ac(Cat.No.25237).Theantigenusedwasapeptidecontainingthehistonemodificationofinterest.Byplottingtheabsorbanceagainsttheantibodydilution(Figure1),thetiteroftheantibodywasestimatedtobe1:2,750.
CrossreactivitytestusingtheantibodydirectedagainstH3K23ac
ADotBlotanalysiswasperformedtotestthecrossreactivityoftheantibodyagainstH3K23ac(Cat.No.25237)withwithpeptidescontainingothermodificationsandunmodifiedsequencesofhistoneH3.Onehundredto0.2pmolofthepeptidecontainingtherespectivehistonemodificationwerespottedonamembrane.Theantibodywasusedatadilutionof1:1,000.Figure2showsahighspecificityoftheantibodyforthemodificationofinterest.
WesternblotanalysisusingtheantibodyagainstH3K23ac
HeLacellsweretreatedwithbutyrateandhistoneextracts(15μg)wereanalysedbyWesternblotusingtheantibodyagainstH3K23ac(Cat.No.25237)diluted1:250inTBS-Tweencontaining5%skimmedmilk(lane1).Lane2showsacoomassiebluestainingofthegel.Thepositionoftheproteinofinterestisindicatedontheleft;theMarker(inkDa)isshownontheright.
Storeat–80°Cforupto2years.Centrifugeafterfirstthawtomaximizeproductrecovery.Aliquottoavoidrepeatedfreeze/thawcycles.Aliquotsmaybestoredat–20°Cforatleastonemonth.