| Assay Method Information | |
| | Characterization of Hits and Compound 15 (MELK-In-1) Derivatives |
| Description: | The top 10 hits from the 1 μM screening and derivatives of MELK-In-1 were subjected to the same assay conditions with varied inhibitor concentrations (0.0005-50 μM) to generate a dose-response curve and IC50. At set time points (0.25, 0.5, 1, 1.5, and 2 min for assays containing 10 nM MELK and 1, 2, 4, 6, and 10 min for assays containing 1 nM MELK), 30 μL aliquots were taken from each reaction and spotted onto 2×2 cm squares of p81 paper. Papers were washed 4 times in 75 mM O-phosphoric acid and once in acetone. Labeled protein was quantified by its associated CPM determined on a Packard 1500 scintillation counter at a sigma value of 2. CPM were translated into nmol 32P incorporated using Equation 1. |
| Affinity data for this assay | |
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