| Assay Method Information | |
| | PDGFR-β Tyrosine Kinase Inhibitory Action |
| Description: | The test substance was prepared with dimethyl sulfoxide (DMSO) to 10 mM and diluted with DMSO so as to reach a concentration of 0.001 to 1000 μM. This DMSO solution was diluted by 8 times with an assay buffer 1 (50 mM HEPES (pH 7.0), 0.02% NaN3, 0.01% Bovine Serum Albumin, 0.1 mM orthovanadate, 1 mM Dithiothreitol, 5 mM MgCl2, and 1 mM MnCl2) and further diluted by 5 times with an assay buffer 2 (50 mM HEPES (pH 7.0), 0.02% NaN3, 0.01% Bovine Serum Albumin, 0.1 mM orthovanadate, 1 mM Dithiothreitol, 5 mM MgCl2, 1 mM MnCl2, and 40 nM Supplemented Enzymatic Buffer (cisbio)).2. Measurement of PDGFR-β Tyrosine Kinase Inhibitory ActionFor the measurement, the HTRF KinEASE-TK kit from Cisbio Bioassays SAS was used. To a 384 well plate, the test substance solution was added at 4 μL each, then 2 μL of a PDGFR-β enzyme solution (final concentration of 1 ng/μL, Carna Biosciences, Inc.) and 4 μL of a substrate solution obtained by adding 0.6 μM ATP to TK substrate-3-biotin (cisbio) were added, and the solution was allowed to react at 30° C. for 30 minutes at a final concentration of the test substance being 0.01 to 10,000 nM.Thereafter, 10 μL of a detection solution (cisbio) was added to each well and allowed to react at 30° C. for 1 hour. The fluorescence intensity was measured with a microplate reader (Spectra Max M5, Molecular device). |
| Affinity data for this assay | |
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