Ubiquitin-AMC is a fluorogenic substrate for a wide range of deubiquitinating enzymes (DUBs), including ubiquitin C-terminal hydrolases (UCHs) and ubiquitin specific proteases (USPs). It is a particularly useful reagent for the study of deubiquitinating activity where detection sensitivity or continuous monitoring of activity is essential.BioMol Online Catalog
Background Ubiquitin-AMC is prepared by the C-terminal derivatization of ubiquitin with 7-amino-4-methylcoumarin and has been shown to be a useful and sensitive fluorogenic substrate for wide range of deubiquitinylating enzymes (DUBs), including ubiquitin C-terminal hydrolases (UCHs) and ubiquitin specific proteases (USPs).
Ubiquitin-AMC has been shown to be a sensitive substrate for UCH-L3 (Km = 0.039 μM) and for Isopeptidase-T (Km = 0.17-1.4 μM),Dang, L.C., Melandri, F.D. and Stein, R.L. Kinetic and mechanistic studies on the hydrolysis of ubiquitin C-terminal 7-amido-4-methylcoumarin by deubiquitinating enzymes. Biochemistry, 37, 1868-1879 (1998) and is particularly useful for studying deubiquitinylating activity where detection sensitivity or continuous monitoring of activity is essential.Mason, D.E., Ek, J., Peters, E.C. and Harris J.L. Substrate profiling of deubiquitin hydrolases with a positional scanning library and mass spectrometry. Biochemistry, 43, 6535-44 (2004)
Typical assay set-up: Assay substrate concentration: 0.01-1.0 μM. Enzyme concentrations, UCH-L3: 10-100pM, isopeptidase-T: 10-100nM. Release of AMC fluorescence by DUB enzymes can be monitored using 380 nm excitation and 460 nm emission wavelengths.
Uses Substrate for deubiquitinylating enzyme activity assays. Identification/confirmation of enzyme deubiquitinylation activity. Investigation of deconjugating enzyme substrate specificity in comparison with alternative UBL-AMC substrates (e.g. NEDD8-AMC)
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