ChIPresultsobtainedwiththeantibodydirectedagainstH4K5,8,12,16ac
ChIPassayswereperformedusinghumanHeLacells,theantibodyagainstH4K5,8,12,16ac(Cat.#25282)andoptimizedPCRprimersetsforqPCR.ChIPwasperformedonshearedchromatinfrom1millioncells.Atitrationoftheantibodyconsistingof1,2,5and10µgperChIPexperimentwasanalyzed.IgG(2µg/IP)wasusedasnegativeIPcontrol.QPCRwasperformedwithprimersforpromoteroftheactivegeneEIF4A2andforaregion1kbupstreamoftheGAPDHgene,usedaspositivecontrols,andfortheinactiveMYOD1geneandtheSat2satelliterepeatregionusedasnegativecontrols.Figure1showstherecovery,expressedasa%ofinput(therelativeamountofimmunoprecipitatedDNAcomparedtoinputDNAafterqPCRanalysis).

ChIP-seqresultsobtainedwiththeantibodydirectedagainstH4K5,8,12,16ac
ChIPwasperformedwith2µgoftheantibodyagainstH4K5,8,12,16ac(Cat.#25282)onshearedchromatinfrom1millionHeLacells.TheimmunoprecipitatedDNAwassubsequentlyanalyzedonanIlluminaGenomeAnalyzer.Librarypreparation,clustergenerationandsequencingwereperformedaccordingtothemanufacturer"sinstructions.The36bptagswerealignedtothehumangenomeusingtheELANDalgorithm.Figure2showsthesignaldistributionalongthecompletelengthofchromosome5(figure2A)andazoomintoa600kbregion(figure2B).Figure2CandDshowtheenrichmentintwogenomicregionsonchromosome3and12,respectively,containingEIF4A2andGAPDHpositivecontrols.ThepositionoftheampliconusedforvalidatingtheQPCRresultsisshownwithanarrow."

Determinationoftheantibodytiter
Todeterminethetiteroftheantibody,anELISAwasperformedusingaserialdilutionoftheantibodydirectedagainstH4K5,8,12,16ac(Cat.#25282)inantigencoatedwells.Theantigenusedwasapeptidecontainingthehistonemodificationofinterest.Byplottingtheabsorbanceagainsttheantibodydilution(Figure3),thetiteroftheantibodywasestimatedtobe1:21,200.

CrossreactivitytestsusingtheantibodydirectedagainstH4K5,8,12,16ac
TotestthecrossreactivityoftheantibodyagainstH4K5,8,12,16ac(Cat.#25282),aDotBlotanalysiswasperformedwithpeptidescontainingotherhistonemodificationsandtheunmodifiedH4.Onehundredto0.2pmoloftherespectivepeptideswerespottedonamembrane.Theantibodywasusedatadilutionof1:20,000.Figure4showsahighspecificityoftheantibodyforthemodificationofinterest.

ImmunofluorescenceusingtheantibodydirectedagainstH4K5,8,12,16ac
MouseNIH3T3cellswerestainedwiththeantibodyagainstH4K5,8,12,16ac(Cat.#25282)andwithDAPI.Cellswerefixedwith4%formaldehydefor10min.andblockedwithPBS/TX-100containing5%normalgoatserumand1%BSA.ThecellswereimmunofluorescentlylabeledwiththeH4K5,8,12,16acantibody(left)diluted1:500inblockingsolutionfollowedbyananti-rabbitantibodyconjugatedtoAlexa488.ThemiddlepanelshowsstainingofthenucleiwithDAPI.Amergeofthetwostainingsisshownontheright.

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