| Assay Method Information | |
| | In Vitro Kinase Assay |
| Description: | Each test was performed twice in a total reaction volume of 30 μL, containing 6 μg of peptide substrate RRRDDDSDDD (New England Biolabs (UK) Ltd., Herts, UK); 10 units of recombinant human CK2 holoenzyme (New England Biolabs); 50 μM ATP and γ-labeled 32P ATP, diluted to specific activity 100 μCi/mM; CK2 buffer (20mM Tris-HCl, pH 7.5; 50mM KCl; 10mM MgCl2) and inhibitor in varying concentrations. Incubation time was 20 min at 30 °C. The reaction was stopped by adding an equal volume of 10% o-phosphoric acid and the reaction mixture was loaded onto 20-mm discs of phosphocellulose paper (Whatman plc, Kent, UK). Discs were washed three times with 1% o-phosphoric acid solution, air-dried at room temperature, and counted by the Cherenkov method in a beta-counter (LKB). As negative control an equal volume of DMSO was added to the reaction mixture. Quercetin, a known CK2 inhibitor, was used as an inhibition positive control. The final concentration of quercetin was 0.55 μM (which inhibited CK2 activity by 50%). |
| Affinity data for this assay | |
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