| Assay Method Information | |
| | Ub-AMC Enzymatic and Kinetic Assays |
| Description: | Full length USP7 was tested for its activity in Ubiquitin-AMC assay in presence or absence of inhibitors. USP7 (5 nM) was pre-incubated for 6 hours at room temperature with different concentrations of inhibitors or DMSO as a control in 50 mM HEPES pH 7.5, 0.5 mM EDTA, 11 μM ovalbumin, and 5 mM DTT. Ubiquitin-AMC (Boston Biochem) was then added to a final concentration of 500 nM. The initial rate of the reaction was measured by collecting fluorescence data at one minute intervals over 30-minute period using a Clariostar fluorescence plate reader at excitation and emission wavelength of 345 and 445 nm respectively. The calculated initial rate values were plotted against inhibitor concentrations to determine IC50s. All the experimental data were plotted using Prism GraphPad. All assays for each compound were performed at least twice for each compound. |
| Affinity data for this assay | |
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