ChIPresultsobtainedwiththeantibodydirectedagainstH2AK5ac
ChIPassayswereperformedusinghumanHeLacells,theantibodyagainstH2AK5ac(Cat.No.25230)andoptimizedPCRprimersetsforqPCR.ChIPwasperformedusingshearedchromatinfrom1.5millioncells.Atitrationoftheantibodyconsistingof0.5,1,2and,5μgperChIPexperimentwasanalysed.IgG(1μg/IP)wasusedasnegativeIPcontrol.QPCRwasperformedwithprimersforaregionapproximately1kbupstreamoftheGAPDHandACTBpromoters,usedaspositivecontrols,andforthecodingregionoftheinactiveMYOD1geneandtheSat2satelliterepeat,usedasnegativecontrols.Figure1showstherecovery,expressedasa%ofinput(therelativeamountofimmunoprecipitatedDNAcomparedtoinputDNAafterqPCRanalysis).

ChIP-seqresultsobtainedwiththeantibodydirectedagainstH2AK5ac
ChIPwasperformedonshearedchromatinfrom1.5millionHeLaS3cellsusing0.5μgoftheantibodyagainstH2AK5ac(Cat.No.25230)asdescribedabove.TheimmunoprecipitatedDNAwassubsequentlyanalysedonanlluminaHiSeq.Librarypreparation,clustergenerationandsequencingwereperformedaccordingtothemanufacturer’sinstructions.The51bptagswerealignedtohehumangenomeusingtheBWAalgorithm.Figure2showstheenrichmentalongthecompletesequenceanda1MbregionoftheX-chromosome(fig2AandB)andingenomicregionsofchromosome7,surroundingtheACTBgene,andofchromosome12,surroundingtheGAPDHgene(fig2CandD).ThepositionoftheampliconusedforChIP-qPCRisindicatedbyanarrow.

Determinationoftheantibodytiter
Todeterminethetiteroftheantibody,anELISAwasperformedusingaserialdilutionoftheantibodydirectedagainstH2AK5ac(Cat.No.25230)inantigencoatedwells.Theantigenusedwasapeptidecontainingthehistonemodificationofinterest.Byplottingtheabsorbanceagainsttheantibodydilution(Figure2),thetiteroftheantibodywasestimatedtobe1:25,000.

CrossreactivitytestsusingtheantibodydirectedagainstH2AK5ac
TotestthecrossreactivityoftheantibodyagainstH2AK5ac(Cat.No.25230),aDotBlotanalysiswasperformedwithpeptidescontainingotherhistonemodificationsandtheunmodifiedH2AK5.Onehundredto0.2pmoloftherespectivepeptideswerespottedonamembrane.Theantibodywasusedatadilutionof1:5,000.Figure3showsahighspecificityoftheantibodyforthemodificationofinterest.

WesternblotanalysisusingtheantibodydirectedagainstH2AK5ac
Westernblotwasperformedonwholecell(25μg,lane1)andhistoneextracts(15μg,lane2)fromHeLacells,andon1μgofrecombinanthistoneH2A,H2B,H3andH4(lane3,4,5and6,respectively)usingtheantibodyagainstH2AK5ac(Cat.No.25230).Theantibodywasdiluted1:1,000inTBS-Tweencontaining5%skimmedmilk.TheMarker(inkDa)isshownontheleft.

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