| Assay Method Information | |
| | USP 36 Inhibition Biochemical Assay Protocol |
| Description: | USP36 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), 3 mM 2-Mercaptoethanol (Sigma, M6250), 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 added to initiate the reaction (2 nM human USP36 (81-461) 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.Fluorescence signal was measured on an EnVision Plate Reader (PerkinElmer) equipped with 485 nm excitation filter and 535 nm emission filters. Measurements were taken at 2.5 minutes intervals for 10 minutes, curves were shown to progress linearly. |
| Affinity data for this assay | |
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