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Bpsbioscience/Anti–H3K9acS10p polyclonal antibody/25267/100 µl
ChIPresultsobtainedwiththeantibodydirectedagainstH3K9acS10p
ChIPassayswereperformedusinghumanosteosarcoma(U2OS)cells,theantibodyagainstH3K9acS10p(Cat.#25267)andoptimizedPCRprimersetsforqPCR.ChIPwasperformedusingshearedchromatinfrom1.5millioncells.Atitrationoftheantibodyconsistingof5and15µlperChIPexperimentwasanalyzed.IgG(5µg/IP)wasusedasnegativeIPcontrol.QPCRwasperformedwithprimersfortheALDOA(fructose-bisphosphatealdolaseA)promoterandforthecodingregionofthemyogenicdifferentiationgene(MYOD),agenethatisinactiveatnormalconditions.Figure1showstherecovery,expressedasa%ofinput(therelativeamountofimmunoprecipitatedDNAcomparedtoinputDNAafterqPCRanalysis).
Determinationofthetiter
Todeterminethetiter,anELISAwasperformedusingaserialdilutionoftheantibodyagainsthumanH3K9acS10p(Cat.#25267).Theantigenusedwasapeptidecontainingthehistonemodificationsofinterest.Byplottingtheabsorbanceagainsttheantibodydilution(Figure2),thetiteroftheantibodywasestimatedtobe1:89,000.
CrossreactivitytestusingtheantibodydirectedagainstH3K9acS10p
ADotBlotanalysiswasperformedtotestthecrossreactivityoftheantibodyagainstH3K9acS10p(Cat.#25267)withpeptidescontainingothermodificationsofhistoneH4andH3orunmodifiedhistoneH3sequences.Onehundredto0.2pmolofthepeptidecontainingtherespectivehistonemodificationwerespottedonamembrane.Theantibodywasusedatadilutionof1:20,000.Figure3showsahighspecificityoftheantibodyforthedoublemodification.
WesternblotanalysisusingtheantibodydirectedagainstH3K9acS10p
HistoneextractsofHeLacells(15µg)wereanalyzedbyWesternblotusingtheantibodyagainstH3K9acS10p(Cat.#25267)diluted1:250inTBS-Tweencontaining5%skimmedmilk.Thepositionoftheproteinofinterestisindicatedontheleft;theMarker(inkDa)isshownontheright.
ImmunofluorescenceusingtheantibodydirectedagainstH3K9acS10p
HeLacellswerestainedwiththeantibodyagainstH3K9acS10p(Cat.#25267)andwithDAPI.Cellswerefixedwith4%formaldehydefor10min.andblockedwithPBS/TX-100containing5%normalgoatserumand1%BSA.ThecellswereimmunofluorescentlylabelledwiththeH3K9acS10pantibody(left)diluted1:500inblockingsolutionfollowedbyananti-rabbitantibodyconjugatedtoAlexa488.ThemiddlepanelshowsstainingofthenucleiwithDAPI.Amergeofthetwostainingsisshownontheright.
Storeat–80°Cforupto2years.Centrifugeafterfirstthawtomaximizeproductrecovery.Aliquottoavoidrepeatedfreeze/thawcycles.Aliquotsmaybestoredat–20°Cforatleastonemonth.
ELISA(1:1000–1:4000)
DB(1:20,000)
WB(1:250)
IF(1:500)
