| Assay Method Information | |
| | In vitro activity assay for USP1 |
| Description: | Each compound to be tested was dissolved with DMSO to 10 mM. The compound and pure DMSO (with a total volume of 50 nL) were loaded to each well of the 384-well plate by using an ECHO instrument to obtain gradient-diluted sample concentrations at different ratios. The enzyme was diluted with a freshly prepared reaction solution (20 mM Tris-HCl (pH 8.0), 2 mM CaCl2, 2 mM β-mercaptoethanol, 0.05% CHAPS, and ddH2O). To each well, 5 μL of diluted enzyme reaction solution was added, then the plate was centrifuged and oscillated to mix the enzyme and the compound, and the mixture was centrifuged again and placed on ice. The kit reporter system and the substrate were diluted with the reaction solution, then 5 μL of diluted liquid was added to each well, and mixed by centrifugation. The mixture was incubated at room temperature for 0.5 h. An Envision plate reader (PerkinElmer, excitation wavelength: 485 nm, emission wavelength: 530 nm) was used to measure the fluorescence signal in each well. The inhibitory activity (IC50 values) of the compound on enzyme activity was calculated by using a four-parameter Logistic Model. |
| Affinity data for this assay | |
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