| Assay Method Information | |
| | Kinase Assay |
| Description: | Recombinant CDK4 and CycD1-GST fusion proteins, purified from baculovirus-infected insect cells (Sf9), were purchased from ProQinase GmbH, Freiburg. CDK4/CycD1 (250 ng/measuring point) was incubated for 3 hours at 22 C. in the presence of different concentrations of test substances (0 uM, and within the range 0.001-10 uM) in 31 ul of assay buffer [50 mM Hepes pH 7.0; 2.5 mM MnCl; 0.05 mM Na ortho-vanadate; 1.0 mM dithiothreitol; 0.25 uM adenosine trisphosphate (ATP); 0.5 uM biotinylated myelin basic protein (bio-MPB, GE Healthcare); 0.05 uCi/measuring point 33P-gamma ATP; 0.005% NP40; 0.025% bovine serum albumin; 3% dimethyl sulphoxide]. The reaction was stopped by adding 50 ul of stop-mix [100 uM ATP; 10 mM EDTA pH 8.0; 0.2% Triton X100; 0.125 mg of streptavidin-SPA Beads (GE Healthcare)]. After incubation for 10 min at room temperature, the SPA beads were pelleted by centrifugation (10 min; 1500 g). |
| Affinity data for this assay | |
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