| Assay Method Information | |
| | USP19 Inhibition Biochemical Assay Protocol |
| Description: | USP19 enzymatic assays were performed in a final volume of 6 μL in buffer containing 20 mM Tris-HCl pH 8.0, (Corning 46-031-CM), and 1 mM GSH (Sigma, G4251), 0.03% BGG (Sigma, G7516), and 0.01% Triton X-100 (Sigma, 93443). Test compounds were serially diluted in DMSO (Sigma, G7516) to obtain 10-point, 3-fold series. Nanoliter quantities were pre-dispensed into 1536 assay plates (Corning, 9110BC) for the concentration response range, 26.6 μM to 1.35 nM. 3 μL of 2× enzyme was added to the assay plates, preincubated with compound for 30 minutes, and then 3 μL of 2× substrate was subsequently added to initiate the reaction (0.3 nM human USP19 (Boston Biochem, E-576) and 25 nM Ub-Rh110MP (UbiQ, UbiQ-126) final concentrations). Enzyme and substrate concentrations and incubation times were optimized for the maximal signal-to-background while maintaining linear initial velocity conditions at a fixed substrate concentration below Km. |
| Affinity data for this assay | |
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