ChIPresultsobtainedwiththeantibodydirectedagainstH3K79me3
ChIPwasperformedwiththeantibodyagainstH3K79me3(Cat.#25263)onshearedchromatinfrom1millionHeLaS3cells.Atitrationoftheantibodyconsistingof1,2,5and10µgperChIPexperimentwasanalyzed.IgG(5µg/IP)wasusedasnegativeIPcontrol.QuantitativePCRwasperformedwithprimersfortheGAPDHpromoterandforexon2oftheinactivemyoglobingene.Figure1showstherecovery,expressedasa%ofinput(therelativeamountofimmunoprecipitatedDNAcomparedtoinputDNAafterqPCRanalysis).TheseresultsareinaccordancewiththeobservationthatH3K79me3showsapreferenceforactivepromoters. 

ChIP-seqresultsobtainedwiththeantibodydirectedagainstH3K79me3
ChIPwasperformedasdescribedaboveusing2µgoftheantibodyagainstH3K79me3(Cat.#25263).TheimmunoprecipitatedDNAwasanalyzedonanIlluminaGenomeAnalyzer.Librarypreparation,clustergenerationandsequencingwereperformedaccordingtothemanufacturer"sinstructions.The36bptagswerealignedtothehumangenomeusingtheELANDalgorithm.Figure2showsthepeakdistributionalongthecompletesequenceanda600kbregionoftheX-chromosome(figure2AandB),ina2Mbregionfromchromosome11(figure2C),andina100kbregionsurroundingtheGAPDHpositivecontrolgene(figure2D). 

Determinationoftheantibodytiter
Todeterminethetiteroftheantibody,anELISAwasperformedusingaserialdilutionofantibodydirectedagainstH3K79me3(Cat.#25263),crudeserumandflow-throughinantigencoatedwells.Theantigenusedwasapeptidecontainingthehistonemodificationofinterest.Byplottingtheabsorbanceagainsttheantibodydilution(Figure3),thetiteroftheantibodywasestimatedtobe1:3,500.

CrossreactivitytestsusingtheantibodydirectedagainstH3K79me3
ADotBlotanalysiswasperformedtotestthecrossreactivityoftheantibodyagainstH3K79me3(Cat.#25263)withpeptidescontainingotherhistonemodificationsandtheunmodifiedH3K79.Onehundredto0.2pmoloftherespectivepeptideswerespottedonamembrane.Theantibodywasusedatadilutionof1:20,000.Figure4showsahighspecificityoftheantibodyforthemodificationofinterest.

WesternblotanalysisusingtheantibodydirectedagainstH3K79me3
HistoneextractsofHeLacells(15µg)wereanalyzedbyWesternblotusingtheantibodyagainstH3K79me3(Cat.#25263),diluted1:1,000inTBS-Tweencontaining5%skimmedmilk.ThemolecularweightMarkerisshownontheright;thelocationoftheproteinofinterestisindicatedontheleft. 

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