| Assay Method Information | |
| | USP9X Enzyme Activity Assay |
| Description: | 1. Test Steps1). A 1× analysis buffer was formulated, consisting of a modified Tris buffer (pH 7.5).2). Compound solutions of different concentrations were formulated with final concentrations of 3,000/1,000/300/100/30/10/3/1 nM (containing 1% DMSO), and added into a 384-well plate.3). A USP9X enzyme solution was formulated with the 1× analysis buffer.4). 10 μL of the USP9X enzyme solution was added into the 384-well plate, and incubated with the compounds at room temperature for 1 h.5). Rhodamine 110 Protein was added into the 1× analysis buffer to formulate a substrate working solution.6). 10 μL of the substrate solution was added into each reaction well, centrifuged for 30 s, and mixed uniformly for 30 s.7). The 384-well plate was placed on a microplate reader for detection, with an excitation wavelength of 480 nm and an emission wavelength of 540 nm. Test was conducted for 30 minutes and the data was collected.8. Fitting the dose-effect curve.PercentageinhibitionrateInh%=(Max-Signal)/(Max-Min)*100. |
| Affinity data for this assay | |
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