ChIPresultsobtainedwiththeantibodydirectedagainstH3K4me3
ChIPassayswereperformedusinghumanHeLacells,theantibodyagainstH3K4me3(Cat.No.25257)andoptimizedPCRprimerpairsforqPCR.ChIPwasperformedusingshearedchromatinfrom1millioncells.Atitrationconsistingof1,2,5and10μgofantibodyperChIPexperimentwasanalyzed.IgG(2μg/IP)wasusedasanegativeIPcontrol.QuantitativePCRwasperformedwithprimersspecificforthepromoteroftheactivegenesGAPDHandEIF4A2,usedaspositivecontrols,andforexon2oftheinactivemyoglobin(MB)geneandtheSat2satelliterepeat,usedasnegativecontrols.Figure1showstherecovery,expressedasa%ofinput(therelativeamountofimmunoprecipitatedDNAcomparedtoinputDNAafterqPCRanalysis).TheseresultsareinaccordancewiththeobservationthattrimethylationofK4athistoneH3isassociatedwiththepromotersofactivegenes.

ChIP-seqresultsobtainedwiththeantibodydirectedagainstH3K4me3
ChIPwasperformedonshearedchromatinfrom1millionHeLacellsusing1μgoftheantibodyagainstH3K4me3(Cat.No.25257)asdescribedabove.TheimmunoprecipitatedDNAwassubsequentlyanalysedonanIlluminaGenomeAnalyzer.Librarypreparation,clustergenerationandsequencingwereperformedaccordingtothemanufacturer’sinstructions.The36bptagswerealignedtothehumangenomeusingtheELANDalgorithm.Figure2showsthepeakdistributionalongthecompletesequenceanda600kbregionoftheX-chromosome(figure2AandB)andintworegionssurroundingtheGAPDHandEIF4A2positivecontrolgenes,respectively(figure2CandD).TheseresultsclearlyshowanenrichmentoftheH3K4trimethylationatthepromotersofactivegenes.

CrossreactivitytestusingtheantibodydirectedagainstH3K4me3
ADotBlotanalysiswasperformedtotestthecrossreactivityoftheantibodyagainstH3K4me3(Cat.No.25257)withpeptidescontainingothermodificationsandunmodifiedsequencesofhistoneH3andH4.Onehundredto0.2pmoloftherespectivepeptideswerespottedonamembrane.Theantibodywasusedatadilutionof1:2,000.Figure3showsahighspecificityoftheantibodyforthemodificationofinterest.

WesternblotanalysisusingtheantibodydirectedagainstH3K4me3
WesternblotwasperformedusinghistoneextractsfromHeLacells(HeLaNE,15μg)andtheantibodyagainstH3K4me3(Cat.No.25257)diluted1:500inTBS-Tweencontaining5%skimmedmilk.AmolecularweightMarker(inkDa)isshownontheleft,thepositionoftheproteinofinterestisshownontheright.

ImmunofluorescenceusingtheantibodydirectedagainstH3K4me3
HeLacellswerestainedwiththeantibodyagainstH3K4me3(Cat.No.25257)andwithDAPI.Cellswerefixedwith4%formaldehydefor10minutesandblockedwithPBS/TX-100containing5%normalgoatserumand1%BSA.ThecellswereimmunofluorescentlylabelledwiththeH3K4me3antibody(left)diluted1:500inblockingsolutionfollowedbyananti-rabbitantibodyconjugatedtoAlexa488.ThemiddlepanelshowsstainingofthenucleiwithDAPI.Amergeofthetwostainingsisshownontheright.

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