| Assay Method Information | |
| | CDK4/Cyclin D1 Mobility Shift Assay |
| Description: | Small molecule inhibition of CDK4/Cyclin D1 kinase activity was evaluated using a fluorescence-based microfluidic mobility shift assay. CDK4/Cyclin D1 catalyzes the production of ADP from ATP during phosphoryl transfer to the substrate peptide, FLPeptide34 (5-FAM-RRRFRPASPLRGPPK-COOH) (Perkin Elmer, 760643). CDK4/Cyclin D1 (Thermo Fisher, PR8064A) at 3 nM was prepared with 10 mM MgCl and 1 M ATP in a buffer containing 40 mM HEPES, 1 mM EGTA, 0.01% Brij-35, 0.05% BSA, and 1 mM DTT and pre-incubated at room temperature for 30 min prior to the start of the reaction. 3 micromolar of the substrate was added to start the reaction. The mobility shift assay electrophoretically separates the fluorescently labeled peptides (substrate and phosphorylated product) following the 30 minute kinase reaction. The reaction was terminated by addition of 0.5 M EDTA. Both substrate and product were measured and the ratio of these values used to generate % conversion of substrate to product by the LabChip EZ reader (Perkin Elmer). IC50 values were calculated using the inhibition of conversion ratio using Dotmatics Knowledge Solutions Studies curve fitting (Dotmatics, Bishops Stortford, UK, CM23). |
| Affinity data for this assay | |
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