CHEMBL5734354

JAK Inhibition Activity Assays: Reagents used in JAK inhibition assay: 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. Required cofactors are added individually to each kinase reaction. Assays were carried out according to vendor's specifications as followings:Prepare indicated substrate in freshly prepared Base Reaction Buffer Deliver any required cofactors to the substrate solution above Deliver indicated kinase into the substrate solution and gently mix Deliver compounds in DMSO into the kinase reaction mixture by Acoustic technology (Echo550; nanoliter range), incubate for 20 minutes at room temperature Deliver 33P-ATP (specific activity 10 μCi/μl) into the reaction mixture to initiate the reaction. Incubate kinase reaction for 2 hours at room temperature Reactions are spotted onto P81 ion exchange paper Detect kinase activity by filter-binding method.