| Assay Method Information | |
| | In Vitro USP1 Kinase Inhibitory Activity |
| Description: | Experimental Methods1) Preparation of 1× assay buffer: 1× assay buffer (modified Tris buffer) was prepared;2) Compound dilution: The compound was transferred to the assay plate by Echo. The final concentration of DMSO is 1%;3) Preparation of enzyme solution: Recombinant human His6-USP1/His6-UAF1 complex protein was added to 1× assay buffer to obtain an enzyme solution;4) Preparation of substrate solution: Ubiquitin rhodamine 110 protein CF (Ub-Rho) was added to 1× assay buffer to obtain a substrate solution;5) 10 μL enzyme solution was transferred to the assay plate, and 10 μL 1× assay buffer was added to the control well;6) The plate was incubated at room temperature for 1 hour;7) 10 μL substrate solution was added to each well to start the reaction, and the plate was centrifuged for 30 seconds, and shaked for 30 seconds;8) The plate was read with Envision after 30 minutes, and the parameters were excitation light 480 nm and emission light 540 nm;9) The data was collected and summarized;10) Curve fitting: The data was fitted in Excel, Inhibition rate formula: Percent inhibition rate=(maximum value−signal value)/(maximum value−minimum value)×100; the data was fitted in XL-Fit,Half inhibition concentration formula: Percent inhibition rate=minimum concentration+(maximum concentration−minimum concentration)/(1+(half inhibition concentration/compound concentration)slope);Half inhibition concentration is referred to as IC50 hereinafter. |
| Affinity data for this assay | |
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