| Assay Method Information | |
| | Experimental Method of USP1 Enzyme Activity |
| Description: | USPli compounds were screened by USP1 enzyme activity detection experiment.1.1 Experimental materials: recombinant human His6-USP1/His6-UAF1 composite protein. (R&D, catalog number E-568-050): Ubiquitin rhodamine 110 (Ub-Rho) (R&D, catalog number U-555-050); Fluorescent 384-well plate (Perkin Elmer, Art. No. 6007279).1.2 Test sample: Compounds of table 3 in this application, whose structural formulae and preparation methods are shown in the above examples.1.3 Experimental Process:(1) Preparing 1× detection buffer (improved Tris buffer, comprising 50 mM Tris-HCl (PH 7.8) (Sigma, article number: T2569-1L), 0.01% Tween-20 (Sigma, article number: P2287-100ML), 1 mM DTT (Sigma, article number: D0632-10G), 0.01% BSA (Sigma, article number: B2064-100G) and 0.5 mM EDTA (Invitrogen, article number: 15575020)).(2) Diluting compounds: preparing a 10 mM (mol/L) solution of the compound to be tested with dimethyl sulfoxide (DMSO, with a purity of 100%); diluting the solution of the compound to be detected in a 3-fold gradient to obtain 10 concentrations, wherein the highest concentration is 10 mM; transferring the diluted solution of the compound to be tested to a fluorescent 384-well plate by Echo acoustic liquid handlers, double holes were set for each concentration, and the final concentration of DMSO is 1 vol %; the final concentrations of the solution of the compounds to be tested is 10000 nM, 3333 nM, 1111 nM, 370 nM, 123 nM, 41 nM, 13.7 nM, 4.6 nM, 1.5 nM and 0.5 nM.(3) Preparing enzyme solution: preparing enzyme solution in 1× detection buffer.(4) Preparing substrate solution: adding Ubiquitin Rhodamine 110 (Ub-Rho) into 1× detection buffer to form the substrate solution.(5) Transferring 10 μL of the enzyme solution prepared in step (3) to the fluorescent 384-well plate.(6) Incubating the resultant for 1 hour at room temperature.(7) Adding 10 UL of the substrate solution prepared in step (4) into each well to start the reaction; wherein the final reaction system composed of 200 nL of the compound to be tested+10 μL of the enzyme solution+10 μL of the substrate solution: the final concentration of enzyme is 0.05 DM, and the final concentration of the substrate solution is 300 nM; and the reaction is carried out with centrifuging for 30s and shaking for 30s.(8) Reading the plate on a multifunctional enzyme-labeling instrument SpectraMax Paradigm for 30 minutes, with an excitation wavelength of 480 nm and an emission wavelength of 540 nm.(9) Collecting data regarding SpectraMax Paradigm.(10) Curve fitting:Using Equation (I) to fit data in Excel to obtain the inhibition value;inhibitionrate%=(maximumsignalvalue-targetsignalvalue)/(maximumsignalvalue-minimumsignalvalue) |
| Affinity data for this assay | |
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