| Assay Method Information | |
| | CDK5 Kinase Inhibition Assay |
| Description: | Human p35/ cyclin dependent kinase 5 (CDK5) was purchased from Millipore Corp. (Bedford, MA), which was expressed as N-terminal GST fusion protein using baculovirus expression system. The assay was conducted in a 96-well plate assay format. The kinase reactions were initiated by addition of ATP- solution (final 500 nM), and were incubated for 45 min at room temperature for CDK5. The reactions were terminated by the addition of Kinase-Glo reagent containing EDTA. Ten minutes after addition of Kinase-Glo reagent, the luminescence was measured on a Wallac ARVO 1420 instrument (PerkinElmer, Shelton, CT). The reaction window was calculated from the difference of the average signals obtained from the control (5% DMSO) and the background wells. The inhibitory activity of compounds is expressed by the inhibitor concentration that produced 50% inhibition (IC50) of the enzyme activity in the absence of inhibitor. The IC50 values were obtained by linear regression analysis with a GraphPad Prism (version 3.02 for Windows, GraphPad software, Inc. San Diego, CA). The best-fit lines were obtained by analyzing the logistic fitting equation. |
| Affinity data for this assay | |
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