| Assay Method Information | |
| | TTK Enzyme Assay |
| Description: | The inhibitory activity of compounds on biochemically purified full-length TTK (Life Technologies, Madison, Wis., U.S.A.) was determined in the IMAP assay (Molecular Devices, Sunnyvale, Calif., U.S.A.). Compounds were dissolved in 100% dimethylsulfoxide (DMSO). At the day of the experiment, the compound stock was diluted in 3.16 fold steps in 100% DMSO, to obtain a 10-point dilution series, followed by further dilution in IMAP reaction buffer, which consists of 10 mM Tris-HCl, pH 7.5, 10 mM MgCl2, 0.01% Tween-20, 0.1% NaN3 and 1 mM freshly prepared dithiothreitol. Compound solution was mixed with an equal volume of full-length TTK enzyme in IMAP reaction buffer. After pre-incubation of 1 hour in the dark at room temperature, fluorescein-labeled MBP-derived substrate peptide (Molecular Devices) was added and ATP to start the reaction. Final enzyme concentration was 3.9 nM, final substrate concentration 50 nM, and final ATP concentration was 5 μM. The reaction was allowed to proceed for 2 hours at room temperature in the dark. The reaction was stopped by quenching with IMAP progressive binding solution according to the protocol of the manufacturer (Molecular Devices). Fluorescein polarization was measured on an Envision multimode reader (Perkin Elmer, Waltham, Mass., USA). Dose-response curves were fitted to a four-parameter logarithmic equation in XLfit 5 (ID Business Solutions, Ltd., Guildford, U.K.). |
| Affinity data for this assay | |
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