Determinationofthetiter
Todeterminethetiter,anELISAwasperformedusingaserialdilutionoftheantibodydirectedagainsthumanJMJD2b(Cat.#25297).Thewellswerecoatedwiththepeptideusedforimmunisationoftherabbit.Byplottingtheabsorbanceagainsttheantibodydilution(Figure1),thetiteroftheantibodywasestimatedtobe1:3,000.
WesternblotanalysisusingtheantibodydirectedagainstJMJD2b
NuclearextractsofHeLacells(40µg)wereanalyzedbyWesternblotusingtheantibodyagainstJMJD2b(Cat.#25297)diluted1:1,000inTBS-Tweencontaining5%skimmedmilk.Thepositionoftheproteinofinterestisindicatedontheright;theMarker(inkDa)isshownontheleft.
ImmunofluorescenceusingtheantibodydirectedagainstJMJD2b
HeLacellswerestainedwiththeantibodyagainstJMJD2b(Cat.No.25297)andwithDAPI.Cellswerefixedwith4%formaldehydefor10minutesandblockedwithPBS/TX-100containing5%normalgoatserumand1%BSA.ThecellswereimmunofluorescentlylabelledwiththeJMJD2bantibody(left)diluted1:200inblockingsolutionfollowedbyananti-rabbitantibodyconjugatedtoAlexa488.ThemiddlepanelshowsstainingofthenucleiwithDAPI.Amergeofthetwostainingsisshownontheright.
Storeat–80°Cforupto2years.Centrifugeafterfirstthawtomaximizeproductrecovery.Aliquottoavoidrepeatedfreeze/thawcycles.Aliquotsmaybestoredat–20°Cforatleastonemonth.
WB(1:1000)
IF(1:200)