| Assay Method Information | |
| | Evaluation of PLK1 Kinase Activity In Vitro |
| Description: | The 33P isotope-labeled kinase activity assay (Reaction Biology Corp) was used to determine the IC50 value to evaluate the inhibitory ability of the test compound to human PLK1 protein kinase.Buffer conditions: 20 mM HEPES (pH 7.5), 10 mM MgCl2, 1 mM EGTA, 0.01% Brij35, 0.02 mg/ml BSA, 0.1 mM Na3VO4, 2 mM DTT, 1% DMSOTest steps: At room temperature, the compounds to be tested were dissolved in DMSO to prepare a 10 mM solution for use. The substrate Casein was dissolved in a freshly prepared buffer (with a final concentration of 20 μM), and the tested PLK1 kinase (with a final concentration of 12 nM) was added thereto and mixed well. The acoustic wave pipetting system Echo 550 was used to add the mother solution of the test compound dissolved in DMSO to the above mixed reaction mixture according to the set final concentration gradient (the highest final concentration was 1 μM, 3-fold dilution, 10 gradients). After incubation at room temperature for 20 minutes, 33P-ATP (30 μM) was added, and after incubation at room temperature for 120 minutes, the reaction mixture was spotted on P81 ion exchange filter paper (Whatman #3698-915). After the filter paper was repeatedly washed with 0.75% phosphoric acid solution, the radioactivity of the phosphorylated substrate remaining on the filter paper was measured. |
| Affinity data for this assay | |
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