| Assay Method Information | |
| | KIF18A Enzyme Assay |
| Description: | KIF18A enzyme assay: The enzymatic activity of KIF18A after treatment with the compounds was measured by an assay of microtubule-stimulated ATPase activity. ADP generated from the ATPase reaction was measured in this assay. The compounds were serially diluted 2-fold in DMSO over a range of 22 concentration points. Recombinant human KIF18A (1-467His-tagged) protein was expressed using a baculovirus system. Concentrations of KIF18A protein, microtubules, and ATP in the reaction were optimized for a standardized homogenous enzyme assay using an ADP-Glo kinase/ATPase assay kit. A reaction buffer [(15 mM Tris, pH 7.5), 10 mM MgCl2, 0.01% PluronicF-68, 1 μM paclitaxel, and 30 μg/mL pig microtubules] was prepared. The compounds and KIF18A protein (30 nM) were added to the prepared reaction buffer, and the reaction mixture was incubated at room temperature for 15 min, followed by addition of ATP (Km, 75 μM). The resulting reaction mixture was incubated at room temperature for another 15 min. 5 μL of ADP-Glo reagent and 2.5 μL of the reaction mixture were mixed and the resulting mixture was incubated at room temperature for 40 min. 10 μL of ADP-Glo detection reagent was added and the mixture was incubated at room temperature for 40 min. Luminescence was measured using a microplate reader and compared with that of the DMSO group, and then the inhibition percentages and IC50 values of the compounds were calculated. |
| Affinity data for this assay | |
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