| Assay Method Information | |
| | CDK4/Cyclin D1 Mobility Shift Assay (MSA) |
| Description: | The purpose CDK4/Cyclin D1 assay is to evaluate the inhibition (% inhibition and IC50 values) in the presence of small molecule inhibitors by using a fluorescence based microfluidic mobility shift assay. CDK4/Cyclin D1 catalyzes the production of ADP from ATP that accompanies the phosphoryl transfer to the substrate peptide 5-FAM-Dyrktide (5-FAM-RRRFRPASPLRGPPK) (Perkin Elmer Peptide 34). The mobility shift assay (MSA) electrophoretically separates the fluorescently labelled peptides (substrate and phosphorylated product) following the kinase reaction. Both substrate and product are measured, and the ratio of these values is used to generate % conversion of substrate to product by the LabChip EZ Reader. Typical reaction solutions contained 2% DMSO (+/− inhibitor), 10 mM MgCl2, 1 mM EGTA, 0.05% BSA, 2 mM DTT, 0.2 mM ATP, 0.01% Brig-35, 1.5 uM 5-FAM-Dyrktide, 2.5 nM CDK4/Cyclin D1 in 50 mM HEPES buffer at pH 7.5. The reaction was initiated with the addition of substrate solution, following a 30-minute pre-incubation of enzyme and inhibitor at 22° C. in the reaction mix. The reaction was stopped after 180 minutes by the addition of 75 uL of 500 mM EDTA and measured on a Perkin Elmer EZ reader instrument. IC50 determinations were made from a plot of the fractional velocity as a function of inhibitor concentration fit to the 4 parameters IC50 equation. |
| Affinity data for this assay | |
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