| Assay Method Information | |
| | In-vitro Enzymatic Activity Assay |
| Description: | Experimental Reagents: Reagent Vendor Cat No.Recombinant Human His6-USP1/His6-UAF1 R&D E-568-050Complex ProteinUbiquitin Rhodamine 110 Protein, CF (Ub-Rho) R&D U-555-050Experimentalcomsumables:Consumables Vendor Cat No.384-Well plate Perkin Elmer 6007279First Experimental Method: 1. Compound Dilution1) The compounds of the present disclosure were prepared to 10 mM with DMSO, and used as test stock solutions.2) The stock solutions of the compounds of the present disclosure were diluted in a 4-fold gradient for 10 concentrations, with a maximum concentration of 10 mM.3) The diluted compounds of the present disclosure were respectively transferred to 384-well plates (diluted by 1,000 folds) by Echo550, 2 multiple wells were set for each concentration, and a final concentration of DMSO was 1%.4) The final concentrations of test compounds were 10,000 nM, 2,500 nM, 625 nM, 156 nM, 39 nM, 9.8 nM, 2.4 nM, 0.61 nM, 0.15 nM and 0.038 nM.2. Enzyme Reaction Experiment1) An enzyme solution was prepared in 1 test buffer.2) Ubiquitin Rhodamine 110 protein, CF (UB-Rho), was added into 1 determination buffer to prepare a substrate solution.3) 10 μL of enzyme solution and 1 reaction buffer were transferred to a 384-well plate, and4) incubated at room temperature for 15 minutes.5) 10 μL of substrate solution was added into each well to start the reaction, centrifuged for 30 seconds, and shaken for 30 seconds.3. Result Detection1) Plate reading was carried out on SpectraMax Paradigm for 30 minutes, with an excitation wavelength of 480 nm and an emission wavelength of 540 nm.2) Data on SpectraMax Paradigm were collected.4. Data analysisAn inhibition (% inh) was calculated by the following formula:inhibition(%)=100% Max-SignalMax-Minwherein, Max represented: a luminous signal intensity of a positive control well without adding the compound;Min represented: a luminous signal intensity of a negative control well without adding the enzyme; andSignal represented: a luminous signal intensity of the compound of the test sample.IC50 was Calculated by the Following Formula:𝑌=Bottom+Top-Bottom1+(IC50 𝑋) HillSlopewherein, Y represented: % inhibition; andX represented: the concentration of the compound. |
| Affinity data for this assay | |
|---|---|
| If you find an error in this entry please send us an E-mail | |