| Assay Method Information | |
| | Biological Assay |
| Description: | An assay for monitoring CDK4/cyclin D1-catalyzed phosphorylation of pRb at the Ser780 site was performed using TR-FRET in a 384-well format, and was used for IC50 determination and kinetic analysis. The reaction was carried out in a 30 uL volume containing 0.3 nM CDK4/cyclin D1, 150 nM biotin-pRb (773-924), 3 uM ATP, and 1.3% DMSO (or compound in DMSO) in the assay buffer (50 mM HEPES-Na, pH 7.5; 5 mM MgCl2, 1 mM DTT, 0.02% Tween-20, and 0.05% BSA). 3 uM ATP was added last to initiate the reaction. The reaction was quenched with 10 uL of 240 mM EDTA-Na (pH 8.0) after 60 min incubation at 22 C. The signal was developed by the addition of 40 uL detection solution containing 40 nM SA-APC, 143 ng/mL anti-phospho-pRb (S780) antibody, and 1 nM Eu-W1024 anti-rabbit IgG antibody in the detection buffer (50 mM HEPES-Na, pH 7.5, 60 mM EDTA-Na, pH 8.0, 0.05% BSA, and 0.1% Triton X-100). After 60 min incubation in the dark, the plate was read on Envision. |
| Affinity data for this assay | |
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