| Assay Method Information | |
| | Kinase Assay |
| Description: | Inhibition of PDGFR-beta kinase activity by compounds disclosed herein was quantified by measuring the amount of [33P] incorporated into the substrate in the presence of a test compound. Briefly, a reaction mixture of 25 uL final volume containing 55 ng of PDGFR-beta kinase (obtained by purifying recombinant N-terminal 6xHis-tagged PDGFR-beta kinase domain construct expressed by baculovirus), 2.5 ug of the substrate [Poly (Glu-Tyr, 4:1), Sigma], kinase reaction buffer (20 mM MOPS pH 7.0, 1 mM EDTA, 5% glycerol, 0.01% Brij-35, 0.1% beta-mercaptoethanol, 1 mg/mL BSA, 2 mM MnCl2, 30 nM ATP, 0.1 uCi per well [33P]-gamma-ATP (2,500-3,000 Ci/mmol)), and a test compound (diluted from 9 uM to the desired final concentration by 4% DMSO) or DMSO (as the control) was incubated at 30C. for 30 minutes. The reaction was stopped by adding 5 uL of 3% phosphoric acid solution. The resultant solution (30 uL) was later harvested by a UniFilter Plate GF/B (PerkinElmer). |
| Affinity data for this assay | |
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