ChIPresultsobtainedwiththeantibodydirectedagainstH3K4me1
ChIPwasperformedwiththeantibodyagainstH3K4me1(Cat.No.25253)onshearedchromatinfrom1millionHeLaS3cells.Atitrationoftheantibodyconsistingof1,2,5and10μgperChIPexperimentwasanalysed.IgG(2μg/IP)wasusedasnegativeIPcontrol.QuantitativePCRwasperformedwithprimersforaregionsurroundingtheACTBandGAS2L1genes,usedaspositivecontrols,andforthepromotersoftheGAPDHandEIF4A2genes,usedasnegativecontrols.Figure1showstherecovery,expressedasa%ofinput(therelativeamountofimmunoprecipitatedDNAcomparedtoinputDNAafterqPCRanalysis).

ChIP-seqresultsobtainedwiththeantibodydirectedagainstH3K4me1
ChIPwasperformedasdescribedabovewith1μgoftheantibodyagainstH3K4me1(Cat.No.25253).TheimmunoprecipitatedDNAwassubsequentlyanalysedonanIlluminaGenomeAnalyzer.Librarypreparation,clustergenerationandsequencingwereperformedaccordingtothemanufacturer’sinstructions.The36bptagswerealignedtothehumangenomeusingtheELANDalgorithm.Figure2AandBshowtheenrichmentinchromosomalregionssurroundingtheACTBandGAS2L1positivecontrolgenes.ThepositionoftheampliconusedintheqPCRvalidationisindicatedbyanarrow.Figure2CandDshowtheH3K4me1signalintwo1Mbregionsofchromosome5andX,respectively.

Determinationofthetiter
Todeterminethetiter,anELISAwasperformedusingaserialdilutionoftheantibodydirectedagainstH3K4me1(Cat.No.25253)inantigencoatedwells.Theantigenusedwasapeptidecontainingthehistonemodificationofinterest.Byplottingtheabsorbanceagainsttheantibodydilution(Figure3),thetiteroftheantibodywasestimatedtobe1:20,100.

CrossreactivitytestsusingtheantibodydirectedagainstH3K4me1
ADotBlotanalysiswasperformedtotestthecrossreactivityoftheantibodyagainstH3K4me1(Cat.No.25253)withpeptidescontainingothermodificationsorunmodifiedsequencesofhistoneH3andH4.Onehundredto0.2pmoloftherespectivepeptideswerespottedonamembrane.Theantibodywasusedatadilutionof1:10,000.Figure4showsahighspecificityoftheantibodyforthemodificationofinterest.

WesternblotanalysisusingtheantibodydirectedagainstH3K4me1
Westernblotwasperformedonwholecell(25μg,lane1)andhistoneextracts(15μg,lane2)fromHeLacells,andon1μgofrecombinanthistoneH2A,H2B,H3andH4(lane3,4,5and6,respectively)usingtheantibodyagainstH3K4me1(Cat.No.25253).Theantibodywasdiluted1:500inTBS-Tweencontaining5%skimmedmilk.Thepositionoftheproteinofinterestisshownontheright,theMarker(inkDa)isshownontheleft.

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