ChIPresultsobtainedwiththeantibodydirectedagainstH4K20me3
ChIPassayswereperformedusinghumanHeLacells,theantibodyagainstH4K20me3(Cat.No.25281)andoptimizedPCRprimersetsforqPCR.ChIPwasperformedusingshearedchromatinfrom1millioncells.Atitrationoftheantibodyconsistingof1,2,5,and10μgperChIPexperimentwasanalysed.IgG(1μg/IP)wasusedasnegativeIPcontrol.QPCRwasperformedwithprimersforpromotersoftheactivegenesc-fosandGAPDH,usedasnegativecontrols,andfortheSat2satelliterepeatregionusedasapositivecontrol.Figure1showstherecovery,expressedasa%ofinput(therelativeamountofimmunoprecipitatedDNAcomparedtoinputDNAafterqPCRanalysis).

ChIP-seqresultsobtainedwiththeantibodydirectedagainstH4K20me3
ChIPwasperformedwith1μgoftheantibodyagainstH4K20me3(cat.No.25281)onshearedchromatinfrom1millionHeLaS3cells.TheimmunoprecipitatedDNAwasanalysedbyQPCRwithoptimizedPCRprimerpairsforthepromoterandcodingregionoftheactiveGAPDHgene,forthecodingregionoftheZNF510geneandfortheSat2satelliterepeat(figure2A).TheimmunoprecipitatedDNAwassubsequentlyanalysedonanIlluminaGenomeAnalyzer.Librarypreparation,clustergenerationandsequencingwereperformedaccordingtothemanufacturer’sinstructions.The36bptagswerealignedtothehumangenomeusingtheELANDalgorithm.Figure2Bshowsthesignaldistributionalongthelongarmofchromosome19andazoomintoanenrichedregioncontainingseveralZNFrepeatgenes.Figure2CandDshowtheenrichmentatZNF12andZNF510onchromosome7and9,respectively.TheseresultsclearlyshowanenrichmentofH4K20me3atZNFrepeatgenes.

Determinationoftheantibodytiter
Todeterminethetiteroftheantibody,anELISAwasperformedusingaserialdilutionoftheantibodydirectedagainstH4K20me3(cat.No.25281),crudeserumandflowthroughinantigencoatedwells.Theantigenusedwasapeptidecontainingthehistonemodificationofinterest.Byplottingtheabsorbanceagainsttheantibodydilution(Figure3),thetiteroftheantibodywasestimatedtobe1:7,400.

CrossreactivitytestusingtheantibodydirectedagainstH4K20me3
ADotBlotanalysiswasperformedtotestthecrossreactivityoftheantibodyagainstH4K20me3(cat.No.25281)withpeptidescontainingotherhistonemodificationsandtheunmodifiedH4K20.Onehundredto0.2pmoloftherespectivepeptideswerespottedonamembrane.Theantibodywasusedatadilutionof1:20,000.Figure4showsahighspecificityoftheantibodyforthemodificationofinterest.

WesternblotanalysisusingtheantibodydirectedagainstH4K20me3
HistoneextractsofHeLacells(15μg)wereanalysedbyWesternblotusingtheantibodyagainstH4K20me3(cat.No.25281)diluted1:1,000inTBS-Tweencontaining5%skimmedmilk.Thepositionoftheproteinofinterestisindicatedontheright;themarker(inkDa)isshownontheleft.

ImmunofluorescenceusingtheantibodydirectedagainstH4K20me3
Humanosteosarcoma(U2OS)cellswerestainedwiththeantibodyagainstH4K20me3(cat.No.25281)andwithDAPI.Cellswerefixedwithicecoldmethanolfor10minutesandblockedwithPBS/TX-100containing5%normalgoatserum.
Figure6A:cellswereimmunofluorescentlylabeledwiththeH4K20me3antibody(left)diluted1:300inblockingsolutionfollowedbyananti-rabbitantibodyconjugatedtoAlexa568orwithDAPI(right),whichspecificallylabelsDNA.
Figure6B:stainingofthecellswiththeH4K20me3antibodyafterincubationoftheantibodywithblockingpeptide(concentration:5ng/μl).

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