ChIPresultsobtainedwiththeantibodydirectedagainstH3S10p
Figure1A:ChIPassayswereperformedusinghumanHeLacellstreatedwithcolcemidtoblockthecellsinmetaphase,theantibodyagainstH3S10p(Cat.#25277)andoptimizedPCRprimersetsforqPCR.ChIPwasperformedonshearedchromatinfrom10,000cells.Atitrationoftheantibodyconsistingof1,2,5,and10µgperChIPexperimentwasanalyzed.IgG(5µg/IP)wasusedasnegativeIPcontrol.QPCRwasperformedwithprimersforthepromoteroftheactivegenesc-fosandRPL30.Figure1Ashowstherecovery,expressedasa%ofinput(therelativeamountofimmunoprecipitatedDNAcomparedtoinputDNAafterqPCRanalysis).
Figure1B:ChIPwasperformedasdescribedaboveusing2µgofH3S10pantibodyandshearedchromatinfrom10,000HeLacellstreatedwithcolcemid(sample1)orfrom10,000untreatedcells(sample2).QPCRwasperformedwithprimersforthepromoteroftheactivegenesc-fosandRPL30,andfortheSat2satelliterepeatregion.

Determinationofthetiter
Todeterminethetiter,anELISAwasperformedusingaserialdilutionoftheantibodydirectedagainstH3S10p(Cat.#25277)andthecrudeserum.Theantigenusedwasapeptidecontainingthehistonemodificationofinterest.Byplottingtheabsorbanceagainsttheantibodydilution(Figure2),thetiteroftheantibodywasestimatedtobe1:5,200.

CrossreactivitytestwiththeantibodydirectedagainstH3S10p
ADotBlotanalysiswasperformedtotestthecrossreactivityoftheantibodyagainstH3S10p(Cat.#25277)withpeptidescontainingothermodificationsofhistoneH3ortheunmodifiedH3S10sequence.Onehundredto0.2pmolofthepeptidecontainingtherespectivehistonemodificationwerespottedonamembrane.Theantibodywasusedatadilutionof1:20,000.Figure3showsahighspecificityoftheantibodyforthemodificationofinterest.NotethattheantibodydoesnotrecognizetheH3S10pmodificationiftheneighboringK9isacetylatedortrimethylated.

WesternblotanalysisusingtheantibodydirectedagainstH3S10p
HeLacellsweretreatedwithcolcemid,whichblocksthecellcycleinmetaphaseand15µgofhistoneextractsofthecellswereanalyzedbyWesternblotusingtheantibodyagainstH3S10p(Cat.#25277)diluted1:1,000inTBS-Tweencontaining5%skimmedmilk.Thepositionoftheproteinofinterestisindicatedontheright;theMarker(inkDa)isshownontheleft.

ImmunofluorescenceusingtheantibodydirectedagainstH3S10p
Humanosteosarcoma(U2OS)cellswerestainedwiththeantibodyagainstH3S10p(Cat.#25277)andwithDAPI.Cellswerefixedwith3.7%formaldehydeinPBSfor20minutesatRT,followedbya20’permeABIlizationwith0.5%TritonX-100inPBSandblockedPBS/TX-100containing5%normalgoatserum.Figure5A:cellswereimmunofluorescentlylabeledwiththeH3S10pantibody(left),diluted1:2,000inblockingbufferfollowedbyananti-rabbitantibodyconjugatedtoAlexa568orwithDAPI(right),whichspecificallylabelsDNA.Figure5B,CandD:stainingofthecellswiththeH3S10pantibodyafterincubationoftheantibodywith2µMblockingpeptidecontainingtheunmodifiedH3S10sequence,thephosphorylatedH3S10andthephosphorylatedH3T11,respectively.

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