| Assay Method Information | |
| | CDKi Biochemical Assay |
| Description: | Biochemical assays measured the inhibitory effects of compounds in this disclosure on the enzymatic activity of CDK enzyme in complex with Cyclin protein partner by phosphorylation of Ser-780 (S780) on retinoblastoma protein peptide (RB1) in the presence of 1 mM adenosine-5-triphosphate (ATP) and varying concentration of test compound in 20 mM 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid (HEPES), pH 7.5, 10 mM MgCl2, 1 mM dithiothreitol (DTT), 0.01% bovine serum albumin (BSA), 0.005% Tween 20. Total Reaction volume of 10 μL proceeded for 60 minutes at room temperature (25° C.) and were quenched with 5 μL of 200 mM 2′,2″,2′″-(Ethane-1,2-diyldinitrilo)tetraacetic acid (EDTA) pH 8.0 before addition of 5 μL detection solution containing 100 nM fluorophore conjugate streptavidin allophycocyanin (SA-APC), 2 nM Europium labelled Anti-p-RB(S780)-K (Perkin Elmer, 64CUSKAY), 50 mM HEPES, pH 7.5, 400 mM potassium fluoride (KF), 0.1% BSA, and 0.01% Tween-20. Phosphorylation of S780 on RB1 peptide (His-MBP-RB1[773-924]—SEQ ID NO. 9) was detected by TR-FRET after 3-hour incubation with detection solution. |
| Affinity data for this assay | |
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