| Assay Method Information | |
| | Protein Kinase Activity Assay |
| Description: | A 20ul mix containing 50mM Tris pH 7.5, 0.05mg/ml BSA, 0.1% β-mercaptoethanol, 10mM MgCl2, 100μM [γ-P32]ATP (5-50cpm/pmol), 0.003% Brij, 150-500ng PDK1 or CaPkh2, and T308tide (from 0.1 to 1mM) was pre-incubated at room temperature (22¿C) for 15 minutes. The kinase reaction was initiated by the addition of the ATP-Mg mixture, followed by the addition of 5μl phosphoric acid (final concentration 0.01%) to stop the reaction. The PDK1 activity assay was performed in a 96 well format and 4μl aliquots spotted on p81 phosphocellulose papers (Whatmann) using ep motion 5070 (Eppendorf), washed in 0.01% phosphoric acid, dried, and then exposed and analyzed using PhophoImager technology (FLA-9000 Starion, Fujifilm). The specific activity of CaPkh2 and PDK1 was estimated using 100μM T308tide. |
| Affinity data for this assay | |
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