| Assay Method Information | |
| | Ubiquitin-Rhodamine110 Assay |
| Description: | USP28 (at 1 nM) was pre-incubated with different concentrations of inhibitors or DMSO as a control in 50 mM Tris pH 8, 50 mM NaCl, 0.002% Tween-20, 5 mM DTT. Compounds and protein were incubated for 30 minutes at room temperature prior to the addition of Ubiquitin-Rho110 (Boston Biochem) substrate for a final concentration of 125 nM. The initial rate of the reaction was measured by collecting fluorescence data at one-minute interval over 30-minute period using a Clariostar fluorescence plate reader at excitation and emission wavelength of 485 nm and 535 nm, respectively. The calculated initial rate values were plotted against inhibitor concentrations to determine IC50s. All the experimental data were plotted using GraphPad Prism . |
| Affinity data for this assay | |
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