ChIPresultsobtainedwiththeantibodydirectedagainstH3K27me1
ChIPassayswereperformedusinghumanHeLacells,theantibodyagainstH3K27me1(Cat.#25240)andoptimizedPCRprimersetsforqPCR.ChIPwasperformedusingshearedchromatinfrom100,000cells.Atitrationoftheantibodyconsistingof1,2,5and10µgperChIPexperimentwasanalyzed.IgG(2µg/IP)wasusedasnegativeIPcontrol.QPCRwasperformedwithprimersforthepromoterandthecodingregionoftheactivegeneGAPDHusedasanegativeandapositivecontroltarget,respectively.Figure1showstherecovery,expressedasa%ofinput(therelativeamountofimmunoprecipitatedDNAcomparedtoinputDNAafterqPCRanalysis).

Determinationoftheantibodytiter
Todeterminethetiteroftheantibody,anELISAwasperformedusingaserialdilutionoftheantibodydirectedagainstH3K27me1(Cat.#25240),crudeserumandflow-through.Theantigenusedwasapeptidecontainingthehistonemodificationofinterest.Byplottingtheabsorbanceagainsttheantibodydilution(Figure1),thetiterofthepurifiedantibodywasestimatedtobe1:32,900.

CrossreactivitytestsusingtheantibodydirectedagainstH3K27me1
ADotBlotanalysiswasperformedtotestthecrossreactivityoftheantibodyagainstH3K27me1(Cat.#25240)withpeptidescontainingothermodificationsandunmodifiedsequencesofhistoneH3andH4.Onehundredto0.2pmolofthepeptidecontainingtherespectivehistonemodificationwerespottedonamembrane.Theantibodywasusedatadilutionof1:20,000.Figure3showsahighspecificityoftheantibodyforthemodificationofinterest.

WesternblotanalysisusingtheantibodydirectedagainstH3K27me1
Histoneextracts(15µg)fromHeLacellswereanalyzedbyWesternblotusingtheantibodyagainstH3K27me1(Cat.#25240)diluted1:1,000inTBS-Tweencontaining5%skimmedmilk.Thepositionoftheproteinofinterestisindicatedontheright;theMarker(inkDa)isshownontheleft.

ImmunofluorescenceusingtheantibodydirectedagainstH3K27me1
Humanosteosarcoma(U2OS)cellswerestainedwiththeantibodyagainstH3K27me1(Cat.#25240)andwithDAPI.Cellswerefixedwith4%formaldehydefor20minutesandblockedwithPBS/TX-100containing5%normalgoatserum.Figure5A:cellswereimmunofluorescentlylabeledwiththeH3K27me1antibody(left)diluted1:1,000inblockingsolutionfollowedbyananti-rabbitantibodyconjugatedtoAlexa568orwithDAPI(right),whichspecificallylabelsDNA.Figure5B,C,DandE:stainingofthecellswiththeH3K27me1antibodyafterincubationoftheantibodywith2ng/µlblockingpeptidecontainingtheunmodifiedandthemono-,di-andtrimethylatedH3K27,respectively.

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