ChIPresultsobtainedwiththeantibodydirectedagainstH4K8ac
ChIPassayswereperformedusinghumanHeLacells,theantibodydirectedagainstH4K8ac(cat.No.25285)andoptimizedPCRprimersetsforqPCR.ChIPwasperformedonshearedchromatinfrom10,000cells.Atitrationoftheantibodyconsistingof1,2,5,and10μgperChIPexperimentwasanalysed.IgG(1μg/IP)wasusedasnegativeIPcontrol.QPCRwasperformedwithprimersforthec-fospromoterandfortheSat2satelliterepeatregion.Figure1showstherecovery,expressedasa%ofinput(therelativeamountofimmunoprecipitatedDNAcomparedtoinputDNAafterqPCRanalysis).

Determinationoftheantibodytiter
Todeterminethetiteroftheantibody,anELISAwasperformedusingaserialdilutionoftheantibodydirectedagainstH4K8ac(cat.No.25285),crudeserumandflowthroughinantigencoatedwells.Theantigenusedwasapeptidecontainingthehistonemodificationofinterest.Byplottingtheabsorbanceagainsttheantibodydilution(Figure2),thetiterofthepurifiedantibodywasestimatedtobe1:16,700.

CrossreactivitytestusingtheantibodydirectedagainstH4K8ac
ADotBlotanalysiswasperformedtotestthecrossreactivityoftheantibodyagainstH4K8ac(cat.No.25285)withpeptidescontainingotherhistonemodificationsandtheunmodifiedH4K8.Onehundredto0.2pmoloftherespectivepeptideswerespottedonamembrane.Theantibodywasusedatadilutionof1:20,000.Figure3showsahighspecificityoftheantibodyforthemodificationofinterest.

WesternblotanalysisusingtheantibodydirectedagainstH4K8ac
HistoneextractsofHeLacells(15μg)wereanalysedbyWesternblotusingtheantibodydirectedagainstH4K8ac(cat.No.25285)diluted1:200inTBS-Tweencontaining5%skimmedmilk.Thepositionoftheproteinofinterestisindicatedontheright;theMarker(inkDa)isshownontheleft.

ImmunofluorescenceusingtheantibodydirectedagainstH4K8ac
MouseNIH3T3cellswerestainedwiththeantibodyagainstH4K8ac(cat.No.25285)andwithDAPI.Cellswerefixedwith4%formaldehydefor10minutesandblockedwithPBS/TX-100containing5%normalgoatserumand1%BSA.ThecellswereimmunofluorescentlylabelledwiththeH4K8acantibody(left)diluted1:500inblockingsolutionfollowedbyananti-rabbitantibodyconjugatedtoAlexa488.ThemiddlepanelshowsstainingofthenucleiwithDAPI.Amergeofthetwostainingsisshownontheright.

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