ChIPresultsobtainedwiththeantibodydirectedagainstH3K4me2
ChIPwasperformedwiththeantibodyagainstH3K4me2(Cat.#25255)onshearedchromatinfrom1millionHeLaS3cells.Atitrationoftheantibodyconsistingof1,2,5and10µgperChIPexperimentwasanalyzed.IgG(2µg/IP)wasusedasnegativeIPcontrol.QuantitativePCRwasperformedwithprimersforthepromoterandcodingregionoftheactiveGAPDHgene,thepromoteroftheactivec-fosgeneandforthecodingregionoftheinactiveTSH2B.Figure1showstherecovery,expressedasa%ofinput(therelativeamountofimmunoprecipitatedDNAcomparedtoinputDNAafterqPCRanalysis).

ChIP-seqresultsobtainedwiththeantibodydirectedagainstH3K4me2
ChIPwasperformedasdescribedaboveusing1µgoftheantibodyagainstH3K4me2(Cat.#25255).TheimmunoprecipitatedDNAwasanalyzedonanIlluminaGenomeAnalyzer.Librarypreparation,clustergenerationandsequencingwereperformedaccordingtothemanufacturer"sinstructions.The36bptagswerealignedtothehumangenomeusingtheELANDalgorithm.Figure2showsthepeakdistributionalongthecompleteX-chromosome(figure2A)andin3chromosomalregionssurroundingtheGAPDH,c-fosandACTBgenes(figure2B,CandD,respectively).

Determinationofthetiter
Todeterminethetiter,anELISAwasperformedusingaserialdilutionoftheantibodydirectedagainstH3K4me2(Cat.#25255),crudeserumandflow-throughinantigencoatedwells.Theantigenusedwasapeptidecontainingthehistonemodificationofinterest.Byplottingtheabsorbanceagainsttheantibodydilution(Figure3),thetiteroftheantibodywasestimatedtobe1:12,600.

CrossreactivitytestusingtheantibodydirectedagainstH3K4me2
ADotBlotanalysiswasperformedtotestthecrossreactivityoftheantibodyagainstH3K4me2(Cat.#25255)withpeptidescontainingothermodificationsofhistoneH3andH4andtheunmodifiedH3K4sequence.Onehundredto0.2pmoloftherespectivepeptideswerespottedonamembrane.Theantibodywasusedatadilutionof1:20,000.Figure4showsahighspecificityoftheantibodyforthemodificationofinterest.

WesternblotanalysisusingtheantibodydirectedagainstH3K4me2
HistoneextractsofHeLacells(15µg)wereanalyzedbyWesternblotusingtheantibodyagainstH3K4me2(Cat.#25255)diluted1:1,000inTBS-Tweencontaining5%skimmedmilk.Thepositionoftheproteinofinterestisindicatedontheright;theMarker(inkDa)isshownontheleft.

ImmunofluorescenceusingtheantibodydirectedagainstH3K4me2
Humanosteosarcoma(U2OS)cellswerestainedwiththeantibodyagainstH3K4me2(Cat.#25255)andwithDAPI.Cellswerefixedwith4%formaldehydefor20minutesandblockedwithPBS/TX-100containing5%normalgoatserum.Figure6A:cellswereimmunofluorescentlylabeledwiththeH3K4me2antibody(left)diluted1:5,000inblockingsolutionfollowedbyananti-rabbitantibodyconjugatedtoAlexa568orwithDAPI(right),whichspecificallylabelsDNA.Figure6B,C,DandE:stainingofthecellswiththeH3K4me2antibodyafterincubationoftheantibodywith5ng/µlblockingpeptidecontainingtheunmodifiedandthemono-,di-andtrimethylatedH3K4,respectively.

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