ChIPresultsobtainedwiththeantibodydirectedagainstH4K5,8,12ac
ChIPassayswereperformedusinghumanK562cells,theantibodyagainstH4K5,8,12ac(Cat.#25283)andoptimizedPCRprimersetsforqPCR.ChIPwasperformedonshearedchromatinfrom100,000cells.Atitrationoftheantibodyconsistingof0.2,0.5,1and2µgperChIPexperimentwasanalyzed.IgG(1µg/IP)wasusedasnegativeIPcontrol.QPCRwasperformedwithprimersforpromoteroftheactivegeneEIF4A2andforaregion1kbupstreamoftheGAPDHgene,usedaspositivecontrols,andfortheinactiveMYOD1geneandtheSat2satelliterepeatregionusedasnegativecontrols.Figure1showstherecovery,expressedasa%ofinput(therelativeamountofimmunoprecipitatedDNAcomparedtoinputDNAafterqPCRanalysis).

ChIP-seqresultsobtainedwiththeantibodydirectedagainstH4K5,8,12ac
ChIPwasperformedwith0.5µgoftheantibodyagainstH4K5,8,12ac(Cat.#25283)onshearedchromatinfrom100,000K562cells.TheimmunoprecipitatedDNAwassubsequentlyanalyzedonanIlluminaGenomeAnalyzer.Librarypreparation,clustergenerationandsequencingwereperformedaccordingtothemanufacturer"sinstructions.The36bptagswerealignedtothehumangenomeusingtheELANDalgorithm.Figure2showsthesignaldistributionalongthecompletelengthofchromosome2(figure2A)andazoomintoa600kbregion(figure2B).Figure2CandDshowtheenrichmentintwogenomicregionsonchromosome3and12,respectively,containingEIF4A2andGAPDHpositivecontrols.

Determinationoftheantibodytiter
Todeterminethetiteroftheantibody,anELISAwasperformedusingaserialdilutionoftheantibodydirectedagainstH4K5,8,12ac(Cat.#25283)inantigencoatedwells.Theantigenusedwasapeptidecontainingthehistonemodificationofinterest.Byplottingtheabsorbanceagainsttheantibodydilution(Figure3),thetiteroftheantibodywasestimatedtobe1:14,500.

CrossreactivitytestsusingtheantibodydirectedagainstH4K5,8,12ac
TotestthecrossreactivityoftheantibodyagainstH4K5,8,12ac(Cat.#25283),aDotBlotanalysiswasperformedwithpeptidescontainingotherhistonemodificationsandtheunmodifiedH4.Onehundredto0.2pmoloftherespectivepeptideswerespottedonamembrane.Theantibodywasusedatadilutionof1:20,000.Figure4showsahighspecificityoftheantibodyforthemodificationofinterest.

WesternblotanalysisusingtheantibodydirectedagainstH4K5,8,12ac
Westernblotwasperformedonwholecell(25µg,lane1)andhistoneextracts(15µg,lane2)fromHeLacells,andon1µgofrecombinanthistoneH2A,H2B,H3andH4(lane3,4,5and6,respectively)usingtheantibodyagainstH4K5,8,12ac(Cat.#25283).Theantibodywasdiluted1:1,000inTBS-Tweencontaining5%skimmedmilk.Thepositionoftheproteinofinterestisindicatedontheright,theMarker(inkDa)isshownontheleft.

ImmunofluorescenceusingtheantibodydirectedagainstH4K5,8,12ac
HeLacellswerestainedwiththeantibodyagainstH4K5,8,12ac(Cat.#25283)andwithDAPI.Cellswerefixedwith4%formaldehydefor10min.andblockedwithPBS/TX-100containing5%normalgoatserumand1%BSA.Figure6A:cellswereimmunofluorescentlylabeledwiththeH4K5,8,12acantibody(left)diluted1:500inblockingsolutionfollowedbyananti-rabbitantibodyconjugatedtoAlexa488.ThemiddlepanelshowsstainingofthenucleiwithDAPI.Amergeofthetwostainingsisshownontheright.Figure6B,C,DandE:stainingofthecellswiththeH4K5,8,12acantibodyafterincubationoftheantibodywith10ng/µlofthefollowingblockingpeptides:H4K5,8,12unmodified(figure6B),H4K5,8,12ac(figure6C),H2A.ZK5,7,11ac(figure6D)andH4K5,8,12,16ac(figure6E).

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