ChIPresultsobtainedwiththemonoclonalantibodydirectedagainstH3K4un
ChIPassayswereperformedusingHeLacells,themonoclonalantibodyagainstH3K4un(Cat.No.25258)andoptimizedPCRprimersetsforqPCR.ChIPwasperformedusingshearedchromatinfrom10⁵cells.Atitrationoftheantibodyconsistingof1,5and10μgperChIPexperimentwasanalysed.IgG(5μg/IP)wasusedasnegativeIPcontrol.QPCRwasperformedwithprimersfortheGAPDHpromoterandforexon2ofthemyoglobingene.Figure1showstherecovery,expressedasa%ofinput(therelativeamountofimmunoprecipitatedDNAcomparedtoinputDNAafterqPCRanalysis).

CrossreactivityofthemonoclonalantibodydirectedagainstH3K4un
TotestthespecificityanELISAwasperformedusingaserialdilutionofthemonoclonalantibodyagainstH3K4un(Cat.No.25258).ThewellswerecoatedwithpeptidescontainingtheunmodifiedH3K4regionaswellasthemono-,di-andtrimethylatedH3K4.Figure2showsahighspecificityoftheantibodyfortheunmodifiedpeptide.

WesternblotanalysisusingthemonoclonalantibodydirectedagainstH3K4un
Histoneextracts(15μg)fromHeLacellswereanalysedbyWesternblotusingthemonoclonalantibodyagainstH3K4un(Cat.No.25258)diluted1:1,000inTBS-Tweencontaining5%skimmedmilk.Thepositionoftheproteinofinterestisindicatedontheright;theMarker(inkDa)isshownontheleft.

ImmunofluorescenceusingthemonoclonalantibodydirectedagainstH3K4un
HeLacellswerestainedwiththeantibodyagainstH3K4un(Cat.No.25258)andwithDAPI.Cellswerefixedwith4%formaldehydefor10minutesandblockedwithPBS/TX-100containing5%normalgoatserumand1%BSA.ThecellswereimmunofluorescentlylabelledwiththeH3K4unantibody(left)diluted1:500inblockingsolutionfollowedbyananti-mouseantibodyconjugatedtoAlexa594.ThemiddlepanelshowsstainingofthenucleiwithDAPI.Amergeofthetwostainingsisshownontheright.

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