ChIPresultsobtainedwiththeantibodydirectedagainstH3K79me2
ChIPassayswereperformedusinghumanHeLacells,theantibodyagainstH3K79me2(Cat.#25262)andoptimizedPCRprimerpairsforqPCR.ChIPwasperformedusingshearedchromatinfrom1millioncells.Atitrationconsistingof1,2,5and10µgofantibodyperChIPexperimentwasanalyzed.IgG(2µg/IP)wasusedasanegativeIPcontrol.QuantitativePCRwasperformedwithprimersspecificforthecodingregionsoftheactivegenesACTBandALDOA,usedaspositivecontrols,andforexon2oftheinactivemyoglobin(MB)geneandtheSat2satelliterepeat,usedasnegativecontrols.Figure1showstherecovery,expressedasa%ofinput(therelativeamountofimmunoprecipitatedDNAcomparedtoinputDNAafterqPCRanalysis).

ChIP-seqresultsobtainedwiththeantibodydirectedagainstH3K79me2
ChIPwasperformedwith1µgoftheantibodyagainstH3K79me2(Cat.#25262)asdescribedaboveandtheimmunoprecipitatedDNAwassubsequentlyanalyzedonanIlluminaGenomeAnalyzer.Librarypreparation,clustergenerationandsequencingwereperformedaccordingtothemanufacturer"sinstructions.The36bptagswerealignedtothehumangenomeusingtheELANDalgorithm.Figure2showsthepeakdistributionalongthecompletesequenceanda1MbregionoftheX-chromosome(figure2AandB),ina100kbregionsurroundingtheACTBpositivecontrolgene(figure2C)andina20kbregionsurroundingtheALDOAgene(figure2D).ThepositionoftheampliconusedforvalidatingtheALDOAenrichmentisindicatedwithanarrow.

Determinationoftheantibodytiter
Todeterminethetiteroftheantibody,anELISAwasperformedusingaserialdilutionoftheantibodydirectedagainsthumanH3K79me2(Cat.#25262),crudeserumandflow-through.Theantigenusedwasapeptidecontainingthehistonemodificationofinterest.Byplottingtheabsorbanceagainsttheantibodydilution(Figure3),thetiteroftheantibodywasestimatedtobe1:8,000.

CrossreactivitytestsusingtheantibodydirectedagainstH3K79me2
ADotBlotanalysiswasperformedtotestthecrossreactivityoftheantibodyagainstH3K79me2(Cat.#25262)withpeptidescontainingothermodificationsandunmodifiedsequencesofhistoneH3.Onehundredto0.2pmolofpeptidecontainingtherespectivehistonemodificationwerespottedonamembrane.Theantibodywasusedatadilutionof1:50,000.Figure4showsahighspecificityoftheantibodyforthemodificationofinterest.

WesternblotanalysisusingtheantibodydirectedagainstH3K79me2
Histoneextracts(15µg)fromHeLacellswereanalyzedbyWesternblotusingtheantibodyagainstH3K79me2(Cat.#25262)diluted1:1,000inTBS-Tweencontaining5%skimmedmilk.Thepositionoftheproteinofinterestisindicatedontheright;theMarker(inkDa)isshownontheleft.

ImmunofluorescenceusingtheantibodydirectedagainstH3K79me2
HeLacellswerestainedwiththeantibodyagainstH3K79me2(Cat.#25262)andwithDAPI.Cellswerefixedwith4%formaldehydefor10min.andblockedwithPBS/TX-100containing5%normalgoatserumand1%BSA.ThecellswereimmunofluorescentlylabelledwiththeH3K79me2antibody(left)diluted1:500inblockingsolutionfollowedbyananti-rabbitantibodyconjugatedtoAlexa488.ThemiddlepanelshowsstainingofthenucleiwithDAPI.Amergeofthetwostainingsisshownontheright. 

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