| Assay Method Information | |
| | Test of Inhibiting NAE Enzyme Activity at the Molecular Level |
| Description: | 1. Preliminary Evaluation Experiment of Inhibiting NAE Enzyme Activity at the Molecular Level by CompoundsHomogeneous Time-Resolved Fluorescence (HTRF) technology was used to detect the in vitro inhibitory effect of the compound on NAE enzyme activity. The experimental operation steps were as follows:(1) the enzyme reaction buffer was prepared using: 50 mM HEPES (pH 7.5), 0.05% BSA, 5 mM MgCl2, 20 μM ATP, 250 μM L-glutathione;(2) a NAE enzyme (human recombinant APPBP1/UBA3) was prepared to 4 nM, and the substrate was prepared to a mixture containing 600 nM His6-NEDD8 and 320 nM GST-UBE2M/Ubc12;(3) the test compound was diluted to 40 μM, and then a 10 times gradient dilution was performed;(4) 10 μL of reaction system was prepared in a 384-well plate, and 5 μL of NAE enzyme, 2.5 μL of mixed substrate and 2.5 μL of test compound were added. The final reaction system included: 2 nM NAE enzyme, 150 nM His6-NEDD8, 80 nM GST-UBE2M/Ubc12 and 10 μM starting test compound. Two repeated wells per group were set up, and a negative control without enzyme and an enzyme group control were additionally set up;(5) the plate was incubated on a shaker at 27° C. for 2 h, then added 10 μL of stop solution: 0.1 M HEPES (pH 7.5), 0.05% Tween 20, 20 mM EDTA, 410 mM KF, Anti-6HIS-Eu Cryptate (CisBio, 1:200), Anti-GST-XL665 (CisBio, 1:200);(6) the plate was left at room temperature overnight, then the plate was read with a fluorescence enzyme-labeled instrument (PE Envision) with excitation light source of Lance and absorption wavelength of 620 nm/665 nm.(7) the inhibition rate of the compound was calculated by the following formula:a. Reading processing: (665/620)*10000 mean value−mean value of negative control groupb.Inhibitionrate(%)=(1-meanvalueofcompoundgroupmeanvalueofenzymecontrolgroup)×100C. IC50 was calculated with a GraphPad software. |
| Affinity data for this assay | |
|---|---|
| If you find an error in this entry please send us an E-mail | |