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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.
Contents
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 = 0.039µM) (Product code UW9745) and for Isopeptidase-T (Km = 0.17-1.4µM) (Product code UW9690), 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.
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)