| Assay Method Information | |
| | Inhibitory Activity on USP1/UAF1 Enzymology |
| Description: | Ubiquitin rhodamine 110 was used as a substrate to detect the activity of the deubiquitinating enzyme USP1/UAF1 after drug treatment. The total test system was 20 μl, and the test buffer comprises 50 mM Tris-HCl (pH 7.8), 0.5 mM EDTA, 0.01% Bovine Serum Albumin, 1 mM DTT, and 0.01% Tween-20. At the beginning of the experiment, 0.5 nM USP1/UAF1 (Bio-Techne, E-568-050) protein was added to a 384-well plate (PerkinElmer, 6008269), and the test compound diluted in a gradient was then added. After incubation at room temperature for 10 min, 500 nM ubiquitin rhodamine 110 (Bio-Techne, U-555-050) was added, and the mixture was incubated at room temperature for 20 min. Enspire™ Multilabel Reader microplate reader (PerkinElmer) was used for detection at excitation light of 485 nm/emission light of 535 nm. |
| Affinity data for this assay | |
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