| Assay Method Information | |
| | Biochemical Kinase Assay Protocol (JAK) |
| Description: | Reagent: Base Reaction buffer; 20 mM Hepes (pH 7.5), 10 mM MgCl2, 1 mM EGTA, 0.02% Brij 35, 0.02 mg/ml BSA, 0.1 mM Na3VO4, 2 mM DTT, 1% DMSO, where required cofactors were added individually to each kinase reaction.Reaction Procedure:1. Prepared indicated substrate in freshly prepared Base Reaction Buffer2. Delivered any required cofactors to the substrate solution above3. Delivered indicated kinase into the substrate solution and gently mix4. Delivered compounds in DMSO into the kinase reaction mixture by Acoustic technology (Echo550; nanoliter range), incubated for 20 minutes at room temperature5. Delivered 33P-ATP into the reaction mixture to initiate the reaction.6. Incubated kinase reaction for 2 hours at room temperature7. Reactions were spotted onto P81 ion exchange paper8. Detected kinase activity by filter-binding method. |
| Affinity data for this assay | |
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