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| | Kinase Inhibition Assay |
| Description: | Preparation of AKT1 and AKT2 and measurement of in vitro inhibitory activity of the above-mentioned compounds against AKT1 and AKT2 kinase activity were carried out with reference to the method disclosed in Biochem. J. Vol. 385, pp 399-408 (2005). In the preparation of AKT1 and AKT2, human AKT1 and AKT2 to which a middle T antigen tag was added were expressed in Sf 9 insect cells, and then AKT1 and AKT2 were prepared following affinity purification and activation by PDK1. The prepared AKT1 and AKT2 were stored at 80C. until the time of measurement of inhibitory activity of the compounds. In the measurement of inhibitory activity of the compounds, AKT1 or AKT2 and each compound of the present invention were preincubated at 25C. for 120 minutes in a buffer solution for reaction (15 mM Tris-HCl pH 7.5, 0.01% Tween-20, 2 mM DTT). As a substrate, biotinylated Crosstide (bioton-KGSGSGRPRTSSFAEG), MgCl2, and ATP were added to final concentrations. |
| Affinity data for this assay | |
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