ChIPresultsobtainedwiththemonoclonalantibodydirectedagainstH4K20me1
ChIPassayswereperformedusinghumanHeLacells,themonclonalantibodyagainstH4K20me1(Cat.#25279)andoptimizedPCRprimersetsforqPCR.ChIPwasperformedonshearedchromatinfrom10,000cells.Atitrationoftheantibodyconsistingof1,2,5,and10µgperChIPexperimentwasanalyzed.IgG(5µg/IP)wasusedasnegativeIPcontrol.QPCRwasperformedwithprimersforthecodingregionoftheactiveGAPDHandtheinactiveTSH2Bgene,fortheGAPDHpromoterandforaregionlocated1kbupstreamoftheGAPDHpromoter.Figure1showstherecovery,expressedasa%ofinput(therelativeamountofimmunoprecipitatedDNAcomparedtoinputDNAafterqPCRanalysis)

ChIP-seqresultsobtainedwiththemonoclonalantibodydirectedagainstH4K20me1
ChIPwasperformedasdescribedaboveusing1µgoftheantibodyagainstH4K20me1(Cat.#25279).TheimmunoprecipitatedDNAwasanalyzedonanIlluminaGenomeAnalyzer.Librarypreparation,clustergenerationandsequencingwereperformedaccordingtothemanufacturer"sinstructions.The36bptagswerealignedtothehumangenomeusingtheELANDalgorithm.Figure2showsthepeakdistributioningenomicregionsfromchromosomes12,7,14and3surroundingtheGAPDH,ACTB,FOSandEIF4A2positivecontrolsgene(figure2A,B,CandD,respectively).

CrossreactivitytestsusingthemonoclonalantibodydirectedagainstH4K20me1
TocheckthespecificityofthemonoclonalantibodyagainstH4K20me1(cat.#25279)aDotBlotwasperformedwithpeptidescontainingdifferentmodificationsofhistoneH3andH4ortheunmodifiedH4K20sequence.Onehundredto0.2pmolofpeptidecontainingtherespectivehistonemodificationwerespottedonamembrane.Theantibodywasusedatadilutionof1:20,000.Figure3showsahighspecificityoftheantibodyforthemodificationofinterest.

WesternblotanalysisusingthemonoclonalantibodydirectedagainstH4K20me1
HistoneextractsofHeLacells(15µg)wereanalyzedbyWesternblotusingthemonoclonalantibodyagainstH4K20me1(cat.#25279)diluted1:200inTBS-Tweencontaining5%skimmedmilk.Thepositionoftheproteinofinterestisindicatedontheright;theMarker(inkDa)isshownontheleft.

ImmunofluorescenceusingthemonoclonalantibodydirectedagainstH4K20me1
Humanosteosarcoma(U2OS)cellswerestainedwiththeantibodyagainstH4K20me1(Cat.#25279)andwithDAPI.Cellswerefixedwithicecoldmethanolfor10min.andblockedwithPBS/TX-100containing5%normalgoatserum.Figure5A:cellswereimmunofluorescentlylabeledwiththeH4K20me1antibody(left),diluted1:600inblockingsolutionfollowedbyananti-mouseantibodyconjugatedtoAlexa488orwithDAPI(right),whichspecificallylabelsDNA.Figure5B:stainingofthecellswiththeH4K20me1antibodyafterincubationoftheantibodywith5ng/µlofthecorrespondingblockingpeptide.

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