Determinationofthetiter
Todeterminethetiteroftheantibody,anELISAwasperformedusingaserialdilutionoftheantibodydirectedagainstH3pan(Cat.#25235).Byplottingtheabsorbanceagainsttheantibodydilution(Figure1),thetiteroftheantibodywasestimatedtobe1:900,000.

CrossreactivitytestusingtheantibodydirectedagainstH3pan
ADotBlotanalysiswasperformedtotestthecrossreactivityoftheantibodyagainstH3pan(Cat.#25235)withthepeptidesusedforimmunizationoftherabbitandotherpeptidescontainingdifferentmodificationsorunmodifiedsequencesofhistoneH3.Onehundredto0.2pmoloftherespectivepeptideswerespottedonamembrane.Theantibodywasusedatadilutionof1:1,000.Figure2showsahighspecificityoftheantibodyforoneoftheunmodifiedpeptides.

WesternblotanalysisusingtheantibodydirectedagainstH3pan
HistoneextractsofHeLacells(15µg)wereanalyzedbyWesternblotusingtheantibodyagainstH3pan(Cat.#25235)diluted1:250inTBS-Tweencontaining5%skimmedmilk(lane1).Lane2showsacoomassiebluestainingofthegel.Thepositionoftheproteinofinterestisindicatedontheleft;theMarker(inkDa)isshownontheright.

ChIPresultsobtainedwiththeantibodydirectedagainstH3pan
ChIPassayswereperformedusinghumanHeLacells,theantibodyagainstH3pan(Cat.#25235)andoptimizedPCRprimersetsforqPCR.ChIPwasperformedonshearedchromatinfrom10,000cellsperIP.Atitrationoftheantibodyconsistingof1,5,and10µlperChIPexperimentwasanalyzed.IgG(5µg/IP)wasusedasnegativeIPcontrol.QPCRwasperformedwithprimersfortheGAPDHpromoter,MYOD,andtheheterochromatinmarkerSat2.Figure4showstherecovery,expressedasa%ofinput(therelativeamountofimmunoprecipitatedDNAcomparedtoinputDNAafterqPCRanalysis).

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