| Assay Method Information | |
| | PLK1 Inhibition Assay |
| Description: | Compounds were screened for their ability to inhibit Plk1 using a radioactive-phosphate incorporation assay. Assays were carried out in a mixture of 25 mM HEPES (pH 7.5), 10 mM MgCl2, 25 mM NaCl, and 2 mM DTT. Final substrate concentrations were 20 μM [γ-33P]ATP (35mCi 33P ATP/mmol ATP, Perkin Elmer/Sigma Chemicals) and 9 uM Sam68 protein. Assays were carried out at room temperature in the presence of 15 nM Plk1. An assay stock buffer solution was prepared containing all of the reagents listed above, with the exception of ATP and the test compound of interest. 0.75 μL of DMSO stock containing serial dilutions of the test compound (typically starting from a final concentration of 10 μM with 2-fold serial dilutions final DMSO concentration 1.5%) was placed in a 384 well plate followed by addition of 25 μL [γ-33P]ATP (final concentration 20 μM). The reaction was initiated by addition of 25 μL of the assay stock buffer solution. The reaction was stopped after 45 minutes by the addition of 25 μL 30% trichloroacetic acid (TCA) containing 10 mM cold ATP. The entire quenched reaction was transferred to a 384-well glass fiber filter plate (Millipore, Cat no. MZFBNOW50). The plate was washed with 3 5% TCA. After drying, 40 μL of Ultima Gold liquid scintillation cocktail (Perkin Elmer) was added to the well prior to scintillation counting in a PerkinElmer TopCount. |
| Affinity data for this assay | |
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