| Assay Method Information | |
| | ASK1 Enzyme Assay |
| Description: | The protein kinase inhibitory activity of the compounds described herein were tested using the ASK1/MAP3K5 assay by Reaction Biology Corp. (Malvern, Pa.). The assay procedure follows (and is also available on the Reaction Biology Corp. website).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;Substrate: 20 μM of myelin basic protein (MBP) and 10 μM ATP;Protein kinase: ASK1/MAP3K5.Reaction Procedure:1. Prepare indicated substrate in freshly prepared Base Reaction Buffer.2. Deliver any required cofactors to the substrate solution.3. Deliver indicated kinase into the substrate solution and gently mix.4. Deliver compounds in DMSO into the kinase reaction mixture by Acoustic; technology (Echo550; nanoliter range), incubate for 20 minutes at room temperature.5. Deliver 33P-ATP (specific activity 10 μCi/μL) into the reaction mixture to initiate the reaction.6. Incubate kinase reaction for 2 hours at room temperature.7. Reactions are spotted onto P81 ion exchange paper.8. Detect kinase activity by filter-binding method. |
| Affinity data for this assay | |
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