| Assay Method Information | |
| | PDGFRbeta Kinase Assay |
| Description: | The kinase used was a recombinant fusion protein composed of N-terminal GST and a C-terminal fragment of human PDGFR (amino acids R561-L1106), expressed in baculovirus-infected insect cells (Sf9) and purified by affinity chromatography (from ProQinase GmbH, Freiburg, Germany). The substrate used for the kinase reaction was biotinylated poly-Glu,Tyr (4:1) copolymer (from Cisbio Bioassays, Codolet, France). For the assay, 50 nl of a 100-fold concentrated solution of the test substance in DMSO was pipetted into a black low-volume 384 well microtitre plate (from Greiner Bio-One, Frickenhausen, Germany), then 2 ul of a solution of PDGFR in assay buffer [50 mM HEPES/NaOH pH 7.5, 10 mM MgCl2, 2.5 mM dithiothreitol, 0.01% (v/v) Triton-X100 (from Sigma)] was added and the mixture was incubated for 15 min, in order to enable preliminary binding of the substance to the enzyme before the kinase reaction. Then the kinase reaction was started by adding 3 ul of a solution of ATP (16.7 uM, final concentration in assay volume 5 ul is 10 uM) and substrate (2.27 ug/ml, final concentration in assay volume 5 ul is 1.36 ug/ml, 30 nM) in assay buffer and the resulting mixture was incubated at 22° C. for 25 min. The PDGFR concentration was adjusted with respect to the respective activity of the enzyme in such a way that the assay proceeded in the linear range (typical enzyme concentration 125 pg/ul). The reaction was stopped by adding 5 ul of a solution of HTRF detection reagents [0.2 uM streptavidin-XL665 (from Cisbio Bioassays, Codolet, France) and 1.4 nM PT66-Eu chelate, a europium chelate-labelled anti-phosphotyrosine antibody (from PerkinElmer), in aqueous EDTA solution (100 mM EDTA, 0.2% (w/v) BSA in 50 mM HEPES/NaOH pH 7.5)]. The resulting mixture was incubated at 22° C. for 1 h to allow formation of a complex of the biotinylated phosphorylated substrate and the detection reagents. Subsequently, the amount of the phosphorylated substrate was evaluated by measuring the resonance energy transfer from the PT66-Eu chelate to the streptavidin-XL665. To this end, the fluorescence emissions at 620 nm and 665 nm after excitation at 350 nm were measured in an HTRF instrument [e.g. PHERAstar (from BMG Labtechnologies, Offenburg, Germany) or ViewLux (from PerkinElmer)]. The ratio of the emissions at 665 nm and 620 nm was taken as a measure of the amount of phosphorylated substrate. The data were normalized (enzyme reaction without inhibitor=0% inhibition; all other assay components except enzyme=100% inhibition). Typically, the test substances were tested on the same microtitre plate at 11 different concentrations in the range from 20 uM to 0.073 nM (20 uM, 5.7 uM, 1.6 uM, 0.47 uM, 0.13 uM, 38 nM, 11 nM, 3.1 nM, 0.89 nM, 0.25 nM and 0.073 nM) in twin values for each concentration. The dilution series were prepared by serial dilutions prior to the assay at the level of the 100-fold concentrated solution (i.e. 2 mM to 7.3 nM in 100% DMSO), although it was possible for the exact concentrations to be different depending on the pipettors used in each case. |
| Affinity data for this assay | |
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