| Assay Method Information | |
| | KIF18A Biochemical Assay |
| Description: | A KIF18A ATPase assay was performed in small-volume, nonbinding, 384-well white plates at a final volume of 10 μL/well. Test compounds (10 mM solution in DMSO; 100 nL/well) were serially diluted 3-fold over 10-point concentration range. A solution of KIF18A (0.4 nM, 5 μL/well; 1-367) in assay buffer (15 mM Tris-HCl [pH 7.5](Boston Bioproducts Inc), 10 mM MgCl2 (Boston Bioproducts Inc), 0.01% Pluronic F-68 (Gibco Inc), 1 uM Taxol (Cytoskeleton Inc), 30 mg/ml pre-formed porcine Microtubules (Cytoskeleton Inc)). The reaction was initiated by the addition of 5 μL of substrate solution (10 μM Ultra-Pure ATP in assay buffer) into the wells. The plates were incubated at room temperature for 45 minutes. After the indicated incubation times, 10 μL ADP-Glo reagent was added to the reactions and the plate was incubated at room temperature for 40 min. Then, 20 μL of kinase detection reagent was added and after an incubation time of 40 min, luminescence was recorded on Envision plate reader (Perkin Elmer, Billerica, MA). |
| Affinity data for this assay | |
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