| Assay Method Information | |
| | KIF18A ADP-Glo Assay |
| Description: | The purpose of KIF18A ADP-Glo assay is to evaluate the inhibition (% inhibition and IC50 values) of small molecule inhibitors by using a Luminescent based ADP-Glo assay. KIF18A catalyzes the production of ADP from ATP. ADP-Glo assay monitors ADP producing biochemical reactions. ADP-Glo is performed in 2 steps upon completion of ATPase reaction: a combined termination of ATPase reaction and depletion of remaining ATP in the first step, and conversion of generated ADP to ATP and the newly produced ATP to light output using luciferase/luciferin reaction in the second step. The luminescent signal generated is proportional to the ADP concentration produced and is correlated with the ATPase activity. KIF18A was purchased from Cytoskeleton (CS—KF18). Typical reaction solutions (5 uL final reaction volume) contained 1% DMSO (±inhibitor), 10 mM MgCI2, 4 uM Taxol, 1000 uM ATP, 0.01% Pluronic F-68, 0.1 mg/ml pre-formed micro-tubules, and 1 nM enzyme complex in 15 mM HEPES buffer at pH 7. 5. The assay was initiated with the addition of ATP solution, following a 30-minute pre-incubation of enzyme and inhibitor at room temperature in the reaction mixture. |
| Affinity data for this assay | |
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