ChIPresultsobtainedwiththeantibodydirectedagainstH4K5ac
ChIPassayswereperformedusinghumanHeLacells,theantibodyagainstH4K5ac(Cat.#25284)andoptimizedPCRprimersetsforqPCR.ChIPwasperformedonshearedchromatinfrom1millioncells.Atitrationoftheantibodyconsistingof1,2,5and10µgperChIPexperimentwasanalyzed.IgG(2µg/IP)wasusedasnegativeIPcontrol.QPCRwasperformedwithprimersforpromoteroftheactivegeneEIF4A2andforaregion1kbupstreamoftheGAPDHgene,usedaspositivecontrols,andfortheinactiveMYOD1geneandtheSat2satelliterepeatregionusedasnegativecontrols.Figure1showstherecovery,expressedasa%ofinput(therelativeamountofimmunoprecipitatedDNAcomparedtoinputDNAafterqPCRanalysis).

ChIP-seqresultsobtainedwiththeantibodydirectedagainstH4K5ac
ChIPwasperformedwith1µgoftheantibodyagainstH4K5ac(Cat.#25284)onshearedchromatinfrom1millionHeLacells.TheimmunoprecipitatedDNAwassubsequentlyanalyzedonanIlluminaGenomeAnalyzer.Librarypreparation,clustergenerationandsequencingwereperformedaccordingtothemanufacturer"sinstructions.The36bptagswerealignedtothehumangenomeusingtheELANDalgorithm.Figure2showsthesignaldistributionalongthecompletelengthofchromosome1(figure2A)andazoomintoa500kbregion(figure2B).Figure2CandDshowtheenrichmentintwogenomicregionsonchromosome3and12,respectively,containingEIF4A2andGAPDHpositivecontrols.ThepositionoftheampliconusedforvalidatingtheQPCRresultsisshownwithanarrow.

Determinationoftheantibodytiter
Todeterminethetiteroftheantibody,anELISAwasperformedusingaserialdilutionoftheantibodydirectedagainstH4K5ac(Cat.#25284)inantigencoatedwells.Theantigenusedwasapeptidecontainingthehistonemodificationofinterest.Byplottingtheabsorbanceagainsttheantibodydilution(Figure3),thetiteroftheantibodywasestimatedtobe1:8,900.

CrossreactivitytestsusingtheantibodydirectedagainstH4K5ac
TotestthecrossreactivityoftheantibodyagainstH4K5ac(Cat.#25284),aDotBlotanalysiswasperformedwithpeptidescontainingotherhistonemodificationsandtheunmodifiedH4.Onehundredto0.2pmoloftherespectivepeptideswerespottedonamembrane.Theantibodywasusedatadilutionof1:5,000.Figure4showsahighspecificityoftheantibodyforthemodificationofinterest.

WesternblotanalysisusingtheantibodydirectedagainstH4K5ac
Westernblotwasperformedonwholecell(25µg,lane1)andhistoneextracts(15µg,lane2)fromHeLacells,andon1µgofrecombinanthistoneH2A,H2B,H3andH4(lane3,4,5and6,respectively)usingtheantibodyagainstH4K5ac(Cat.#25284).Theantibodywasdiluted1:500inTBS-Tweencontaining5%skimmedmilk.TheMarker(inkDa)isshownontheleft. 

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