ChIPresultsobtainedwiththeantibodydirectedagainstH3K27ac
ChIPassayswereperformedusingHeLacells,theantibodyagainstH3K27ac(Cat.No.25239)andoptimizedPCRprimersetsforqPCR.ChIPwasperformedusingshearedchromatinfrom1millioncells.Atitrationoftheantibodyconsistingof1,2,5and10μgperChIPexperimentwasanalysed.IgG(2μg/IP)wasusedasnegativeIPcontrol.QuantitativePCRwasperformedwithprimersforthepromoteroftheactiveACTBandEIF4A2genes,usedaspositivecontrols,andforthecodingregionoftheinactiveMYT1andTSH2Bgenes,usedasnegativecontrols.Figure1showstherecovery,expressedasa%ofinput(therelativeamountofimmunoprecipitatedDNAcomparedtoinputDNAafterqPCRanalysis).TheseresultsareinaccordancewiththeobservationthatH3K27acetylationisenrichedatthepromotersofactivegenes.

ChIP-seqresultsobtainedwiththeantibodydirectedagainstH3K27ac
ChIPwasperformedonshearedchromatinfrom1millionHeLaS3cellsusing2μgoftheantibodyagainstH3K27ac(Cat.No.25239)asdescribedabove.QuantitativePCRwasperformedwithprimersforthepromoteroftheactiveACTBandEIF4A2genes,usedaspositivecontrols,andforthecodingregionoftheinactiveMYT1andMBgenes,usedasnegativecontrols(Figure2A).TheimmunoprecipitatedDNAwassubsequentlyanalysedonanIlluminaGenomeAnalyzer.Librarypreparation,clustergenerationandsequencingwereperformedaccordingtothemanufacturer’sinstructions.The36bptagswerealignedtothehumangenomeusingtheELANDalgorithm.Figure2BshowsthepeakdistributionalongthecompleteX-chromosomeandintworegionssurroundingtheACTBandEIF4A2positivecontrolgenes,respectively(figure2CandD).

Determinationoftheantibodytiter
Todeterminethetiteroftheantibody,anELISAwasperformedusingaserialdilutionoftheantibodydirectedagainstH3K27ac(Cat.No.25239)inantigencoatedwells.Theantigenusedwasapeptidecontainingthehistonemodificationofinterest.Byplottingtheabsorbanceagainsttheantibodydilution(Figure3),thetiteroftheantibodywasestimatedtobe1:4,000.

DotblotanalysisusingtheantibodydirectedagainstH3K27ac
ADotBlotanalysiswasperformedtotestthecrossreactivityoftheantibodyagainstH3K27ac(Cat.No.25239)withpeptidescontainingotherhistonemodificationsandtheunmodifiedH3K27sequence.Onehundredto0.2pmoloftherespectivepeptideswerespottedonamembrane.Theantibodywasusedatadilutionof1:25,000.Figure4showsahighspecificityoftheantibodyforthemodificationofinterest.

WesternblotanalysisusingtheantibodydirectedagainstH3K27ac
Westernblotwasperformedonwholecell(40μg,lane1)andhistoneextracts(15μg,lane2)fromHeLacells,andon1μgofrecombinanthistoneH2A,H2B,H3andH4(lane3,4,5and6,respectively)usingtheantibodyagainstH3K27ac(Cat.No.25239).Theantibodywasdiluted1:1,000inTBS-Tweencontaining5%skimmedmilk.Thepositionoftheproteinofinterestisindicatedontheright;theMarker(inkDa)isshownontheleft.

ImmunofluorescenceusingtheantibodydirectedagainstH3K27ac
HeLacellswerestainedwiththeantibodyagainstH3K27ac(Cat.No.25239)andwithDAPI.Cellswerefixedwith4%formaldehydefor10minutesandblockedwithPBS/TX-100containing5%normalgoatserumand1%BSA.ThecellswereimmunofluorescentlylabelledwiththeH3K27acantibody(left)diluted1:500inblockingsolutionfollowedbyananti-rabbitantibodyconjugatedtoAlexa488.ThemiddlepanelshowsstainingofthenucleiwithDAPI.Amergeofthetwostainingsisshownontheright.

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