ChIPresultsobtainedwiththeantibodydirectedagainstH3K9/14ac
ChIPassayswereperformedusingHeLacells,theantibodyagainstH3K9/14ac(Cat.No.25264)andoptimizedprimerpairsforqPCR.ChIPwasperformedusingshearedchromatinfrom1.5millioncells.Atitrationoftheantibodyconsistingof1,2,5and10μgperChIPexperimentwasanalysed.IgG(5μg/IP)wasusedasnegativeIPcontrol.QPCRwasperformedusingprimersspecificforthepromoteroftheACTBgeneasapositivecontroltargetandforexon2oftheMBgeneasanegativecontroltarget.Figure1showstherecovery(therelativeamountofimmunoprecipitatedDNAcomparedtoinputDNA).TheseresultsconfirmtheobservationthatacetylationofH3K9/14ispresentatactivepromoters.

ChIP-seqresultsobtainedwiththeantibodydirectedagainstH3K9/14ac
ChIPwasperformedwith1μgoftheantibodyagainstH3K9/14ac(Cat.No.25264)onshearedchromatinfrom1millionHeLaS3cells.IgG(2μg/IP)wasusedasanegativeIPcontrol.TheimmunoprecipitatedDNAwasanalysedbyQPCRwithoptimizedPCRprimerpairsforthepromotersoftheactiveGAPDHandc-fosgenes,usedaspositivecontroltargets,andthecodingregionoftheinactiveMBgeneandtheSat2satelliterepeat,usedasnegativecontroltargets(figure2A).TheimmunoprecipitatedDNAwassubsequentlyanalysedwithanIlluminaGenomeAnalyzer.Librarypreparation,clustergenerationandsequencingwereperformedaccordingtothemanufacturer’sinstructions.The36bptagswerealignedtothehumangenomeusingtheELANDalgorithm.Figure2showsthepeakdistributionalongthecompletesequenceanda800kbregionoftheX-chromosome(figure2BandC)andin100kbregionssurroundingtheRBM3,GAPDHandc-fosgenes(figure2D,EandF).TheseresultsclearlyshowanenrichmentoftheH3K9/14doubleacetylationatthepromotersofactivegenes.

Determinationoftheantibodytiter
Todeterminethetiteroftheantibody,anELISAwasperformedusingaserialdilutionoftheantibodydirectedagainstH3K9/14ac(Cat.No.25264),crudeserumandflowthroughinantigencoatedwells.Theantigenusedwasapeptidecontainingthehistonemodificationofinterest.Byplottingtheabsorbanceagainsttheantibodydilution(Figure3),thetiterofthepurifiedantibodywasestimatedtobe1:5,900.

CrossreactivitytestsusingtheantibodydirectedagainstH3K9/14ac
ADotBlotanalysiswasperformedtotestthecrossreactivityoftheantibodyagainstH3K9/14ac(Cat.No.25264)withpeptidescontainingotherhistonemodificationsandtheunmodifiedH3K9/14sequence.Onehundredto0.2pmoloftherespectivepeptideswerespottedonamembrane.Theantibodywasusedatadilutionof1:20,000.Figure4showsahighspecificityoftheantibodyforthemodificationofinterest.

WesternblotanalysisusingtheantibodydirectedagainstH3K9/14ac
HistoneextractsofHeLacells(15μg)wereanalysedbyWesternblotusingtheantibodydirectedagainstH3K9/14ac(Cat.No.25264)diluted1:1,000inTBS-Tweencontaining5%skimmedmilk.Thepositionoftheproteinofinterestisindicatedontheright;theMarker(inkDa)isshownontheleft.

ImmunofluorescenceusingtheantibodydirectedagainstH3K9/14ac
MouseNIH3T3cellswerestainedwiththeantibodyagainstH3K9/14ac(Cat.No.25264)andwithDAPI.Cellswerefixedwith4%formaldehydefor10minutesandblockedwithPBS/TX-100containing5%normalgoatserumand1%BSA.ThecellswereimmunofluorescentlylabelledwiththeH3K9/14acantibody(left)diluted1:500inblockingsolutionfollowedbyananti-rabbitantibodyconjugatedtoAlexa488.ThemiddlepanelshowsstainingofthenucleiwithDAPI.Amergeofthetwostainingsisshownontheright.

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