| Assay Method Information | |
| | Kinase Assay |
| Description: | The substrate DYRKtide (synthetic peptide RRRFRPASPLRGPPK) was dissolved in freshly prepared Base Reaction Buffer (20 mM Hepes (pH 7.5), 10 mM MgCl2, 1 mM EGTA, 0.02% Brij35, 0.02 mg/ml BSA, 0.1 mM Na3VO4, 2 mM DTT, 1% DMSO) at a concentration of 20 μM. DYRK1B was added to the substrate solution in a concentration of 0.3 nM and gently mixed. Dilution series of the compounds according to the present invention in DMSO were prepared. Each dilution was added to a batch of the above reaction mix, followed 20 min later by addition of a mixture of ATP and 33P ATP (specific activity 0.01 μCi/μl final) to a final concentration of 10 μM. Reactions were carried out at 25° C. for 120 min, followed by spotting the reactions onto P81 ion exchange filter paper. Unbound phosphate was removed by extensive washing of the filters in 0.75% phosphoric acid. After subtraction of background derived from control reactions containing inactive enzyme, kinase activity data were expressed as the percent remaining kinase activity in test samples compared to vehicle (DMSO) reactions. |
| Affinity data for this assay | |
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