ChIPresultsobtainedwiththemonoclonalantibodydirectedagainstH3K9un
ChIPassayswereperformedusinghumanHeLacells,themonclonalantibodyagainstH3K9un(Cat.No.25274)andoptimizedPCRprimersetsforqPCR.ChIPwasperformedonshearedchromatinfrom1millioncells.Atitrationoftheantibodyconsistingof1,2,5,and10μgperChIPexperimentwasanalysed.IgG(2μg/IP)wasusedasnegativeIPcontrol.QPCRwasperformedwithprimersforthecodingregionsoftheMYOD1andMBgenesandforaregion1kbupstreamoftheGAPDHpromoter,usedaspositivecontrols,andfortheZNF510codingregion,theEIF4A2promoterandtheSat2satelliterepeatregion,usedasnegativecontrols.Figure1showstherecovery,expressedasa%ofinput(therelativeamountofimmunoprecipitatedDNAcomparedtoinputDNAafterqPCRanalysis).

WesternblotanalysisusingthemonoclonalantibodydirectedagainstH3K9un
Westernblotwasperformedonwholecell(25μg,lane1)andhistoneextracts(15μg,lane2)fromHeLacells,andon1μgofrecombinanthistoneH2A,H2B,H3andH4(lane3,4,5and6,respectively)usingtheantibodyagainstH3K9un(Cat.No.25274).Theantibodywasdiluted1:1,000inTBS-Tweencontaining5%skimmedmilk.Thepositionoftheproteinofinterestisindicatedontheright;theMarker(inkDa)isshownontheleft.

ImmunofluorescenceusingthemonoclonalantibodydirectedagainstH3K9un
HeLacellswerestainedwiththeantibodyagainstH3K9un(cat.No.25274)andwithDAPI.Cellswerefixedwith4%formaldehydefor10minutesandblockedwithPBS/TX-100containing5%normalgoatserumand1%BSA.ThecellswereimmunofluorescentlylabelledwiththeH3K9unantibody(left)diluted1:500inblockingsolutionfollowedbyananti-mouseantibodyconjugatedtoAlexa594.ThemiddlepanelshowsstainingofthenucleiwithDAPI.Amergeofthetwostainingsisshownontheright.

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