ChIPresultsobtainedwiththeantibodydirectedagainstH3K36me1
ChIPassayswereperformedusinghumanU2OScells,theantibodyagainstH3K36me1(Cat.#25246)andoptimizedPCRprimerpairsforqPCR.ChIPwasperformedusingshearedchromatinfrom1.5millioncellsandstringentwashingconditions.Atitrationconsistingof1,2,5and10µgofantibodyperChIPexperimentwasanalyzed.IgG(1µg/IP)wasusedasanegativeIPcontrol.QuantitativePCRwasperformedwithprimersforthepromoteroftheconstitutivelyexpressedGAPDHgeneandfortheinactivegeneMYOD.Figure1showstherecovery,expressedasa%ofinput(therelativeamountofimmunoprecipitatedDNAcomparedtoinputDNAafterqPCRanalysis).

Determinationoftheantibodytiter
Todeterminethetiteroftheantibody,anELISAwasperformedusingaserialdilutionoftheantibodydirectedagainstH3K36me1(Cat.#25246),crudeserumandflow-through.Theantigenusedwasapeptidecontainingthehistonemodificationofinterest.Byplottingtheabsorbanceagainsttheantibodydilution(Figure2),thetiteroftheantibodywasestimatedtobe1:46,000.

CrossreactivitytestsusingtheantibodydirectedagainstH3K36me1
ADotBlotanalysiswasperformedtotestthecrossreactivityoftheantibodyagainstH3K36me1(Cat.#25246)withpeptidescontainingothermodificationsofhistoneH3ortheunmodifiedH3K36.Onehundredto0.2pmolofthepeptidecontainingtherespectivehistonemodificationwerespottedonamembrane.Theantibodywasusedatadilutionof1:20,000.Figure3showsahighspecificityoftheantibodyforthemodificationofinterest.

WesternblotanalysisusingtheantibodydirectedagainstH3K36me1
Histoneextracts(15µg)fromHeLacellswereanalyzedbyWesternblotusingtheantibodyagainstH3K36me1(Cat.#25246)diluted1:1,000inTBS-Tweencontaining5%skimmedmilk.Thepositionoftheproteinofinterestisindicatedontheright;theMarker(inkDa)isshownontheleft.

ImmunofluorescenceusingtheantibodydirectedagainstH3K36me1
Humanosteosarcoma(U2OS)cellswerestainedwiththeantibodyagainstH3K36me1(Cat.#25246)andwithDAPI.Cellswerefixedwith4%formaldehydefor20minutesandblockedwithPBS/TX-100containing5%normalgoatserum.Figure5A:cellswereimmunofluorescentlylabeledwiththeH3K36me1antibody(left)diluted1:500inblockingsolutionfollowedbyananti-rabbitantibodyconjugatedtoAlexa568orwithDAPI(right),whichspecificallylabelsDNA.Figure5B,C,DandE:stainingofthecellswiththeH3K36me1antibodyafterincubationoftheantibodywith2ng/µlblockingpeptidecontainingtheunmodifiedandthemono-,di-andtrimethylatedH3K36,respectively.

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