| Assay Method Information | |
| | Inhibitor Selectivity Assay |
| Description: | The fluorogenic substrate used in this study was ubiquitin-AMC (Boston Biochem). All assays were performed in 384-well black plates (Corning) in a total volume of 24 μL of the assay buffer containing 50 mM HEPES (pH 7.5), 5 mM DTT, 0.1 mg mL−1 BSA, and 0.01% Triton X-100 (v/v) in triplicate. A series of compound concentrations (0 to 200 μM final concentration at 2-fold serial dilution) in 100%DMSO was prepared in a 384-well plate. Then 3× compound solutions were prepared in the assay buffer prior to assays. A total of 8 μL of each enzyme solution was distributed into wells, and 8 μL of varying concentrations of compounds was added and incubated for 10 min. The enzyme reaction was initiated by adding 8 μL of thesubstrate (50 μM final concentration), and fluorescence intensity was continuously monitored at excitation/emission wavelengths of 350 nm/460 nm for 10 min. |
| Affinity data for this assay | |
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