| Assay Method Information | |
| | Inhibition of the Kinesin Spindle Protein KSP/Eg5 |
| Description: | The motor domain of the human kinesin spindle protein KSP/Eg5 (from tebu-bio/Cytoskeleton Inc, No. 027EG01-XL) is incubated at a concentration of 10 nM with 50 μg/ml taxol- (from Sigma No. T7191-5MG) stabilized microtubuli (bovine or porcine, from tebu-bio/Cytoskeleton Inc) for 5 min at RT in 15 mM PIPES, pH 6.8 (5 mM MgCl2 and 10 mM DTT, from Sigma). The freshly prepared mixture was aliquoted into a 384-well MTP. The inhibitors to be examined at concentrations of 1.0×10−6 M to 1.0×10−13 M and ATP (final concentration 500 μM, from Sigma) were then added. Incubation was carried out at RT for 2 h. ATPase activity was detected by detecting the inorganic phosphate formed using malachite green (from Biomol). After addition of the reagent, the assay was incubated at RT for 50 minutes prior to detection of the absorption at a wavelength of 620 nm. Monastrol (Fa. Sigma, M8515-1 mg) and Ispinesib (from Adooq A10486) were used as positive control. The individual data of the dose-activity curve are octuple determinations. The IC50 values are means of three independent experiments. The 100% control was the sample which had not been treated with inhibitors. |
| Affinity data for this assay | |
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