ChIPresultsobtainedwiththemonoclonalantibodydirectedagainstH3K4me3
ChIPassayswereperformedusinghumanHeLacells,themonoclonalantibodyagainstH3K4me3(Cat.No.25256)andoptimizedPCRprimerpairsforqPCR.ChIPwasperformedusingshearedchromatinfrom1millioncells.Atitrationconsistingof1,2,5and10μgofantibodyperChIPexperimentwasanalyzed.IgG(2μg/IP)wasusedasanegativeIPcontrol.QuantitativePCRwasperformedwithprimersspecificforthepromoteroftheconstitutivelyexpressedGAPDHandc-fosgenes,usedaspositivecontrols,andforexon2oftheinactivemyoglobin(MB)geneandtheSat2satelliterepeat,usedasnegativecontrols.Figure1showstherecovery,expressedasa%ofinput(therelativeamountofimmunoprecipitatedDNAcomparedtoinputDNAafterqPCRanalysis).TheseresultsareinaccordancewiththeobservationthattrimethylationofK4athistoneH3isassociatedwiththepromotersofactivegenes.

ChIP-seqresultsobtainedwiththemonoclonalantibodydirectedagainstH3K4me3
ChIPwasperformedonshearedchromatinfrom1millionHeLaS3cellsusing2μgoftheantibodyagainstH3K4me3(Cat.No.25256)asdescribedabove.TheimmunoprecipitatedDNAwassubsequentlyanalysedonanIlluminaGenomeAnalyzer.Librarypreparation,clustergenerationandsequencingwereperformedaccordingtothemanufacturer’sinstructions.The36bptagswerealignedtothehumangenomeusingtheELANDalgorithm.Figure2showsthepeakdistributionalongtwo5Mbregionsofchromosome3and5(figure2AandB,respectively)andintwo100kbregionssurroundingtheGAPDHandc-fospositivecontrolgenes(figure2CandD).TheseresultsclearlyshowanenrichmentoftheH3K4trimethylationatthepromotersofactivegenes.

CrossreactivityofthemonoclonalantibodydirectedagainstH3K4me3
TotestthespecificityanELISAwasperformedusingaserialdilutionofthemonoclonalantibodyagainstH3K4me3(Cat.No.25256).ThewellswerecoatedwithpeptidescontainingtheunmodifiedH3K4aswellasthemono-,di-andtrimethylatedH3K4andthetrimethylatedH3K9.Figure3showsahighspecificityoftheantibodyforthemodificationofinterest.

WesternblotanalysisusingthemonoclonalantibodydirectedagainstH3K4me3
Histoneextracts(15μg)fromHeLacellswereanalysedbyWesternblotusingthemonoclonalantibodyagainstH3K4me3(Cat.No.25256)diluted1:1,000inTBS-Tweencontaining5%skimmedmilk.Thepositionoftheproteinofinterestisindicatedontheright;theMarker(inkDa)isshownontheleft.

ImmunofluorescenceusingthemonoclonalantibodydirectedagainstH3K4me3
HeLacellswerestainedwiththeantibodyagainstH3K4me3(Cat.No.25256)andwithDAPI.Cellswerefixedwith4%formaldehydefor10minutesandblockedwithPBS/TX-100containing5%normalgoatserumand1%BSA.ThecellswereimmunofluorescentlylabelledwiththeH3K4me3antibody(left)diluted1:500inblockingsolutionfollowedbyananti-mouseantibodyconjugatedtoAlexa594.ThemiddlepanelshowsstainingofthenucleiwithDAPI.Amergeofthetwostainingsisshownontheright.

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