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 = 0.039µM) and for Isopeptidase-T (Km = 0.17-1.4µM), and is particularly useful for studying deubiquitinylating activity where detection sensitivity or continuous monitoring of activity is essential.
Typical assay set-up: Assay substrate concentration: 0.01-1.0µM.  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.