| Assay Method Information | |
| | Inhibition Assay |
| Description: | Human PLK1 (Millipore, catalog number 14-777M) (7.2 nM) in 20 mM Hepes, pH 7.6, 10 mM MgCl2, 1 mM EDTA, 0.2 mg/mL BSA, and 100 μM ATP was pre-incubated with inhibitors (8-10 concentrations, in duplicate) for 30 min at room temperature. Kinase reactions were initiated by the addition of 0.4 μCi/μL of γ-32P-ATP (6000 Ci/mmol, NEN) and 0.5 mg/mL dephosphorylated α-casein (Sigma) and incubated for 30 min at room temperature. Kinase activity was determined by spotting 5 μL of each reaction onto dried sheets of nitrocellulose pre-washed with 1M NaCl in 0.1% H3PO4. After blotting each kinase reaction, the nitrocellulose sheets were washed once with 1% AcOH solution. Kinase activity was determined by spotting 5 μL of each reaction onto dried sheets of nitrocellulose that had been pre-washed with 1 M NaCl in 0.1% H3PO4. The nitrocellulose sheets were washed once with 1% AcOH solution and twice with a solution of 1 M NaCl in 0.1% H3PO4 (5-10 minutes per wash). |
| Affinity data for this assay | |
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