| Assay Method Information | |
| | Assay on Inhibition of KIF18A Enzymatic Activity |
| Description: | The reaction system consisted of a test compound, the recombinant protein KIF18A (aa 1-467), ATP (Promega Inc), and an assay buffer. The test compound was prepared into a 0.5 mM stock solution using DMSO (Sigma Inc) and then serially diluted in DMSO. The assay buffer consisted of an aqueous solution of 15 mM Tris, 10 mM MgCl2 (Sigma Inc), 0.01% Pluronic F-68 (Life Technologies Inc), 1 μM paclitaxel (Cytoskeleton Inc), 30 μg/mL porcine tubulin (Cytoskeleton Inc), and 2% DMSO. The KIF18A protein (final concentration: 80 nM) and the compound at different concentrations (1 μL) were added to the prepared assay buffer (50 μL), and the mixture was incubated at room temperature for 15 min. Then, ATP (final concentration: 80 μM) was added to the reaction mixture, and the resulting mixture was incubated at room temperature for 3 h. After the reaction was completed, 5 μL of the ADP-Glo™ reagent and 2.5 μL of the reaction mixture were added to a 384-well plate (Grenier Inc). The resulting mixture was mixed homogeneously, then sealed by an aluminum foil sealing film, and incubated at room temperature in the dark for 40 min. Finally, 10 μL of the ADP-GloTM assay reagent was added to each reaction well, and the mixture was incubated at room temperature in the dark for 40 min. After all reactions were completed, the luminescence values of the wells were read on a microplate reader (Molecular Device_SpectraMax Id5), and the inhibition ratios were calculated. |
| Affinity data for this assay | |
|---|---|
| If you find an error in this entry please send us an E-mail | |