| Assay Method Information | |
| | Fluorometric Ub-AMC Hydrolysis Assay |
| Description: | Ubiquitin-7-amino-4-methylcoumarin (Ub-AMC) was generated as previously described.(7) The enzymatic reaction was conducted in fluorometric assay buffer (50 mM HEPES (pH 7.5), 0.5 mM EDTA, 1 mM DTT, and 0.1 mg/mL bovine serum albumin (BSA) (RPI Corporation, Mount Prospect, IL)) at 25°C to determine in vitro IC50 values. 2 nM UCHL1, UCHL5, USP15, USP2, or 2 pM UCHL3 were incubated with increasing concentrations of inhibitor in assay buffer for 5 min at room temperature. To initiate the reaction, 1 uM Ub-AMC was added and the fluorescence signal was detected for 60 s with a 380 nm excitation and 460 nm emission filter set. The data was normalized against a DMSO control, which was treated as 100% enzyme activity, and normalized data was fit to afour-parameter equation with variable slope using GraphPad Prism (La Jolla, CA) to obtain IC50. |
| Affinity data for this assay | |
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