| Assay Method Information | |
| | In Vitro Activity Assay |
| Description: | DYRK1A assay. Substrate, HT-PRD (Proline rich domain, residues 746-864 of dynamin 1a, prepared as N-terminal tagged 6×His fusion protein), was diluted in dilution buffer (25 mM Tris-HCl, pH 7.4 and 100 mM NaCl) to a concentration of 2 ng/μl or higher and used to coat a 96-well plate (BD Falcon #353072) with 100 μl per well (200 ng/well unless otherwise indicated) at 4° C. overnight. Unbound materials were washed away with dilution buffer and wells were blocked with 150 μl blocking buffer (2% BSA, 1×PBS, and 0.25% Tween 20) at room temperature for 60 min. After blocking, wells were washed extensively with dilution buffer before subjecting to phosphorylation. DYRK1A phosphorylation was performed in wells with 100 μl reaction mix containing 25 mM HEPES, pH7.4, 100 mM NaCl, 5 mM MgCl2, 100 UM ATP (Sigma-Aldrich Chemicals), inhibitor if needed, and 5 ng HT-497 (6×His tagged rat truncated DYRK1A isoform X1 containing residues 1-497). Reactions were initiated by adding HT-497 and continued for 30 min (unless otherwise indicated) at 30° C. At the end point, wells were washed with 350 μl dilution buffer three times to terminate the reaction. A set of inhibition experiments typically consisted of a no-inhibitor control plus a series of eight inhibitor concentrations in the range of 0.000625 UM-100 μM (final) depending on the strength of inhibitor. |
| Affinity data for this assay | |
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