ChIP-seqresultsobtainedwiththeantibodydirectedagainstH3K36me3
ChIPwasperformedwith2μgoftheantibodyagainstH3K36me3(Cat.No.25250)onshearedchromatinfrom1millionHeLaS3cells.IgG(2μg/IP)wasusedasanegativeIPcontrol.TheimmunoprecipitatedDNAwasanalysedbyQPCRwithoptimizedPCRprimerpairsforthepromoterandcodingregionoftheactiveGAPDH,foraregionlocated1kbupstreamoftheGAPDHpromoterandforthecodingregionoftheactiveACTBgene(figure1A).TheimmunoprecipitatedDNAwassubsequentlyanalysedonanIlluminaGenomeAnalyzer.Librarypreparation,clustergenerationandsequencingwereperformedaccordingtothemanufacturer’sinstructions.The36bptagswerealignedtothehumangenomeusingtheELANDalgorithm.Figure1Bshowstheobtainedprofilesingenomicregionsofchromosome12(includingtheGAPDHpositivecontrol),7(includingtheACTBpositivecontrol),14and3,respectively.TheseresultsclearlyshowanenrichmentoftheH3K36me3atactivegenes.

ChIPresultsobtainedwiththeantibodydirectedagainstH3K36me3
ChIPassayswereperformedusingHeLacells,theantibodyagainstH3K36me3(Cat.No.25250)andoptimizedPCRprimersetsforqPCR.ChIPwasperformedusingshearedchromatinfrom1millioncells.Atitrationconsistingof1,2,5and10μgofantibodyperChIPexperimentwasanalyzed.IgG(2μg/IP)wasusedasanegativeIPcontrol.QPCRwasperformedwithprimersforthepromoterandcodingregionoftheactiveGAPDH,foraregionlocated1kbupstreamoftheGAPDHpromoterandfortheSat2satelliterepeat.Figure2showstherecovery,expressedasa%ofinput(therelativeamountofimmunoprecipitatedDNAcomparedtoinputDNAafterqPCRanalysis).

Determinationoftheantibodytiter
Todeterminethetiteroftheantibody,anELISAwasperformedusingaserialdilutionoftheantibodydirectedagainstH3K36me3(Cat.No.25250)andthecrudeserum.Theantigenusedwasapeptidecontainingthehistonemodificationofinterest.Byplottingtheabsorbanceagainsttheantibodydilution(Figure3),thetiterofthepurifiedantibodywasestimatedtobe1:19,300.

CrossreactivitytestsusingtheantibodydirectedagainstH3K36me3
ADotBlotanalysiswasperformedtotestthecrossreactivityoftheantibodyagainstH3K36me3(Cat.No.25250)withpeptidescontainingotherH3andH4modificationsandtheunmodifiedsequence.Onehundredto0.2pmolofpeptidecontainingtherespectivehistonemodificationwerespottedonamembrane.Theantibodywasusedatadilutionof1:20,000.Figure4showsahighspecificityoftheantibodyforthemodificationofinterest.

WesternblotanalysisusingtheantibodydirectedagainstH3K36me3
Histoneextracts(15μg)fromHeLacellswereanalysedbyWesternblotusingtheantibodydirectedagainstH3K36me3(Cat.No.25250)diluted1:1,000inTBS-Tweencontaining5%skimmedmilk.Thepositionoftheproteinofinterestisindicatedontheright;theMarker(inkDa)isshownontheleft.

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