Ubiquitin7-amido-4-methylcoumarin(Ub-AMC)isasensitivefluorescence-basedsubstrateofdeubiquitinatingenzymes.Itisusefulformonitoringdeubiquitinatingenzymes’activityorscreeninginhibitorsofdeubiquitinatingenzymes.TheconcentrationofUb-AMCusedindeubiquitinatingreactionsisusually0.1-1µM.ThereleasedAMCfluorescencecanbemonitoredusingafluorometerorplatereaderwithexcitationandemissionwavelengthsat360nmand460nm,respectively.
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Coomassie-stainedSDS-PAGE
5µgpurifiedUb-AMC
Thereactioncontained2nMbovine26Sproteasomeand1mMUb-AMC.AMCfluorescencewasmonitoredbyaplatereaderusingexcitationandemissionfiltersetat360/40and460/30nm,respectively.