ChIPresultsobtainedwiththeantibodydirectedagainstH3K36me2
ChIPassayswereperformedusinghumanHeLacells,theantibodyagainstH3K36me2(Cat.#25248)andoptimizedPCRprimersetsforqPCR.ChIPwasperformedusingshearedchromatinfrom10,000cells.Atitrationoftheantibodyconsistingof1,5,and10µlperChIPexperimentwasanalyzed.Additionally,thesametitrationwasanalyzedafterincubationoftheantibodywith5nmolofthecorrespondingblockingpeptidefor1houratroomtemperature.IgG(5µg/IP)wasusedasnegativeIPcontrol.QPCRwasperformedwithprimersforthepromoteroftheactivegenesGAPDHandALDOAandforthecodingregionofthemyogenicdifferentiationgene(MYOD).Figure1showstherecovery,expressedasa%ofinput(therelativeamountofimmunoprecipitatedDNAcomparedtoinputDNAafterqPCRanalysis).

ChIP-seqresultsobtainedwiththeantibodydirectedagainstH3K36me2
ChIPwasperformedwith0.5µloftheantibodyagainstH3K36me2(Cat.#25248)onshearedchromatinfrom1millionHeLacells.TheimmunoprecipitatedDNAwassubsequentlyanalyzedonanIlluminaGenomeAnalyzer.Librarypreparation,clustergenerationandsequencingwereperformedaccordingtothemanufacturer"sinstructions.The36bptagswerealignedtothehumangenomeusingtheELANDalgorithm.Figure2showsthesignaldistributionalong3genomicregionsofchromosome20,12andX,respectively.

Determinationofthetiter
Todeterminethetiter,anELISAwasperformedusingaserialdilutionoftheantibodydirectedagainstH3K36me2(Cat.#25248).Theantigenusedwasapeptidecontainingthehistonemodificationofinterest.Byplottingtheabsorbanceagainsttheantibodydilution(Figure3),thetiteroftheantibodywasestimatedtobe1:31,000.

CrossreactivitytestusingtheantibodydirectedagainstH3K36me2
AdotblotanalysiswasperformedtotestthecrossreactivityoftheantibodyagainstH3K36me2(Cat.#25248)withpeptidescontainingothermodificationsandunmodifiedsequencesofhistoneH3.Onehundredto0.2pmolofthepeptidecontainingtherespectivehistonemodificationwerespottedonamembrane.Theantibodywasusedatadilutionof1:100,000.Figure4showsahighspecificityoftheantibodyforthemodificationofinterest.

WesternblotanalysisusingtheantibodydirectedagainstH3K36me2
HistoneextractsofHeLacells(15µg)wereanalyzedbyWesternblotusingtheantibodyagainstH3K36me2(Cat.#25248)diluted1:1,000inTBS-Tweencontaining5%skimmedmilk.Thepositionoftheproteinofinterestisindicatedontheright;theMarker(inkDa)isshownontheleft.TheresultoftheWesternanalysiswiththeantibodyisshowninlane1;lane2showsthesameanalysisafterincubationoftheantibodywith5nmolofthecorrespondingblockingpeptidefor1houratroomtemperature.

ImmunofluorescenceusingtheantibodydirectedagainstH3K36me2
HeLacellswerestainedwiththeantibodyagainstH3K36me2(Cat.#25248)andwithDAPI.Cellswerefixedwith4%formaldehydefor10min.andblockedwithPBS/TX-100containing5%normalgoatserumand1%BSA.ThecellswereimmunofluorescentlylabelledwiththeH3K36me2antibody(left)diluted1:500inblockingsolutionfollowedbyananti-rabbitantibodyconjugatedtoAlexa488.ThemiddlepanelshowsstainingofthenucleiwithDAPI.Amergeofthetwostainingsisshownontheright. 

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