| Assay Method Information | |
| | Biochemical Assay for Inhibitors of CK2α Kinase Activity Assay |
| Description: | Final assay conditions comprised 0.2 nM CK2α, 50 μM peptide substrate (RRRADDSDDDDD), 15 μM ATP in 1× reaction buffer (40 mM Tris pH7.5, 200 mM NaCl, 20 mM MgCl2, 0.1 mg/mL BSA, 1% DMSO). The assay was conducted as follows:1. Appropriate serial dilutions of test compound were prepared using Echo (Labcyte) and 50 nL of 100× compound in 100% DMSO transferred to the assay plate (white opaque OptiPlate-384, Perkin-Elmer).2. Enzyme and peptide substrate were prepared in fresh reaction buffer and added to the assay plate in a total volume of 3 μl and incubated at room temperature for 15 minutes.3. 2 μL of ATP solution freshly prepared in reaction buffer was added to start the reaction.4. After 120 minutes, the reaction was stopped by addition of 5 μl ADP-Glo reagent (Promega V9102) and the plate incubated at room temperature for a further 60 minutes.5. 10 μL of Kinase Detection reagent (Promega V9102) was added to assay plate and incubated for a further 30 minutes prior to reading luminescence on an Envision (Perkin-Elmer).Data was analysed to calculate compound IC50 and Ki as follows:1. All assay plates contained 32 wells designated as 0% inhibition control wells, which were treated with vehicle only (1% DMSO) and 32 wells designated as 100% inhibition control wells, which were treated with a high concentration of non-specific kinase inhibitor in 1% DMSO.2. Percent inhibition in each test well was calculated using the formula (MEAN0% inhibition control wells−test well reading)/(MEAN0% inhibition control wells−MEAN100% inhibition control wells)×100%.3. IC50 was determined using a standard 4-parameter fit method (Model 205, XL-fit).4. Percent activity was calculated for each well using: (Test well reading−MEAN100% inhibition control wells)/(MEAN0% inhibition control wells−MEAN100% inhibition control wells).5. Morrison Ki was determined using Morrison Ki equation (XL-fit).Assay 2: Biochemical Assay for Inhibitors of CLK2 Kinase Activity |
| Affinity data for this assay | |
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