| Assay Method Information | |
| | Radioactive Assay |
| Description: | The capacity to inhibit the kinase activity of the enzyme is estimated by measuring the residual kinase activity of the enzyme in the presence of different concentrations of the test compound (generally from 0.17 to 10 000 nM). A dose-response curve is produced, which allows an IC50 (50% inhibitory concentration) to be determined. The kinase activity is measured by a radioactive assay of the amount of radioactive phosphate (33P) incorporated into a fragment of the protein NuMA (Nuclear Mitotic Apparatus protein) after 30 minutes of incubation at 37 ° C. The test compound is first dissolved at different concentrations in dimethyl sulphoxide (DMS (J). Reaction takes place in the wells of a FlashPlate microtiter plate (Nickel Chelate FlashPlate-96, PerkinElmer). Each well (100 l) contains 10 nM Aurora A, 500 nM NuMA, 1 M ATP and 0.2 Ci ATP- -33P in a buffer of 50 mM Tris-HCl, pH=7.5; 10 mM MgCl2; 50 mM NaCl, 1 mM dithiothreitol. The final percentage of DMSO is 3%. |
| Affinity data for this assay | |
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