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Assay Method Information |
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| | Kinase Inhibiting Activity Assay |
| Description: | 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 Reaction Procedure: (i) Prepare indicated substrate in freshly prepared Base Reaction Buffer (ii) Deliver any required cofactors to the substrate solution above (iii) Deliver indicated kinase into the substrate solution and gently mix (iv) Deliver compounds in DMSO into the kinase reaction mixture (v) Deliver 33P-ATP (specific activity 0.01 μCi/μl final) into the reaction mixture to initiate the reaction. (vi) Incubate kinase reaction for 120 minutes at room temperature (vii) Reactions are spotted onto P81 ion exchange paper (Whatman #3698-915) (viii) Wash filters extensively in 0.1% phosphoric acid. (ix) Dry filters and measure counts in scintillation counter Kinase Information: CHK-1H Genbank Accession # AF016582 Recombinant full length construct, N-terminal GST tagged, purified from insect cells. No special measures were taken to activate this kinase. Final concentration in assay=0.5 nM Substrate: CHKtide Peptide sequence: [KKKVSRSGLYRSPSMPENLNRPR] Final concentration in assay=20 μM No additional cofactors are added to the reaction mixture |
| Affinity data for this assay | |
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