Determinationoftheantibodytiter
Todeterminethetiteroftheantibody,anELISAwasperformedusingaserialdilutionofthemonoclonalantibodyagainstH2BS14p(Cat.No.25234).Theantigenusedwasapeptidecontainingthehistonemodificationofinterest.Byplottingtheabsorbanceagainsttheantibodydilution(Figure1),thetiteroftheantibodywasestimatedtobe1:2,000.

CrossreactivitytestsusingthemonoclonalantibodyagainstH2BS14p
TotestthecrossreactivityoftheantibodyagainstH2BS14p(Cat.No.25234),aDotBlotanalysiswasperformedwithpeptidescontainingotherhistonephosphorylationsandtheunmodifiedH2BS14.Onehundredto0.2pmoloftherespectivepeptideswerespottedonamembrane.Theantibodywasusedatadilutionof1:2,000.Figure2showsahighspecificityoftheantibodyforthemodificationofinterest.

WesternblotanalysisusingtheantibodydirectedagainstH2BS14p
HeLacellsweretreatedwithcolcemidtoblockthecellcycleinmetaphaseand15μgofhistoneextractsofthesecellswereanalysedbyWesternblotusingtheantibodyagainstH2BS14p(cat.No.25234)diluted1:200inTBS-Tweencontaining5%skimmedmilk.Thepositionoftheproteinofinterestisindicatedontheright;theMarker(inkDa)isshownontheleft.

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