ChIPresultsobtainedwiththeantibodydirectedagainstH3K79me1
ChIPassayswereperformedusinghumanHeLacells,theantibodyagainstH3K79me1(Cat.#25261)andoptimizedPCRprimerpairsforqPCR.ChIPwasperformedusingshearedchromatinfrom1millioncells.Atitrationconsistingof1,2,5and10µgofantibodyperChIPexperimentwasanalyzed.IgG(2µg/IP)wasusedasanegativeIPcontrol.QuantitativePCRwasperformedwithprimersspecificforaregion1kbupstreamofthepromoterandthecodingregionoftheactiveGAPDHgene,usedaspositivecontrols,andforexon2oftheinactivemyoglobin(MB)geneandtheSat2satelliterepeat,usedasnegativecontrols.Figure1showstherecovery,expressedasa%ofinput(therelativeamountofimmunoprecipitatedDNAcomparedtoinputDNAafterqPCRanalysis).

ChIP-seqresultsobtainedwiththeantibodydirectedagainstH3K79me1
ChIPwasperformedwith1µgoftheantibodyagainstH3K79me1(Cat.#25261)asdescribedaboveandtheimmunoprecipitatedDNAwassubsequentlyanalyzedonanIlluminaGenomeAnalyzer.Librarypreparation,clustergenerationandsequencingwereperformedaccordingtothemanufacturer"sinstructions.The36bptagswerealignedtothehumangenomeusingtheELANDalgorithm.Figure2showsthepeakdistributionalongthecompletesequenceanda1MbregionoftheX-chromosome(figure2AandB),in100kbregionssurroundingtheGAPDHpositivecontrolandEIF4A2genes(figure2CandD).

Determinationoftheantibodytiter
Todeterminethetiteroftheantibody,anELISAwasperformedusingaserialdilutionoftheantibodydirectedagainstH3K79me1(Cat.#25261).Theantigenusedwasapeptidecontainingthehistonemodificationofinterest.Byplottingtheabsorbanceagainsttheantibodydilution(Figure3),thetiteroftheantibodywasestimatedtobe1:11,500.

CrossreactivitytestusingtheantibodydirectedagainstH3K79me1
ADotBlotanalysiswasperformedtotestthecrossreactivityoftheantibodyagainstH3K79me1(Cat.#25261)withpeptidescontainingotherhistonemodificationsandtheunmodifiedH3K79.Onehundredto0.2pmoloftherespectivepeptideswerespottedonamembrane.Theantibodywasusedatadilutionof1:20,000.Figure4showsahighspecificityoftheantibodyforthemodificationofinterest.

WesternblotanalysisusingtheantibodydirectedagainstH3K79me1
Westernblotwasperformedonwholecell(25µg,lane1)andhistoneextracts(15µg,lane2)fromHeLacells,andon1µgofrecombinanthistoneH3(lane3)usingtheantibodyagainstH3K79me1(Cat.#25261).Theantibodywasdiluted1:200inTBS-Tweencontaining5%skimmedmilk.Thepositionoftheproteinofinterestisindicatedontheright;theMarker(inkDa)isshownontheleft. 

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