Assay Method Information
Assay Name:  VEGFR2 Activity Inhibition Assay
Description:  The in vitro activity of VEGFR2 was determined by detecting the phosphorylation level of the substrate in the kinase reaction using a homogeneous time-resolved fluorescence (HTRF) kinase detection kit (Cisbio, catalog number 62TKOPEC). The reaction buffer contains an enzyme reaction buffer (1×) provided in the kit, 5 mM MgCl2, 1 mM MnCl2, 1 mM DTT, 0.01% BSA and 0.005% Tween 20. The humanized recombinant VEGFR2 protein (Cat. No. 10012-H20B1) was purchased from Sino Biological Inc., and diluted to a 0.3 ng/μL kinase solution with the reaction buffer. The substrate reaction solution includes 0.3 μM biotin-labeled tyrosine kinase substrate and 3.5 μM ATP which were diluted with the reaction buffer. The assay buffer includes 0.1 ng/μL Eu3+ labeled cage antibody and 18.75 nM streptavidin labeled XL665 (Cisbio, catalog number 610SAXLB) which were diluted with the reaction buffer.The compound was dissolved and diluted to 10 μM in 100% DMSO, then a 4-fold serial dilution was performed with DMSO to the lowest concentration of 0.61 nM, and each concentration point was diluted 40-fold with the reaction buffer.4 μL of Compound solution and 2 μL of VEGFR2 kinase solution were added to a 384-well detection plate (Corning, catalog number 4512), mixed uniformly and incubated at room temperature for 15 minutes. Then 4 μL of the substrate reaction solution was added, and the reaction mixture was incubated at room temperature for 30 minutes. Then 10 μL of the assay buffer equal to the volume of the reaction was added, mixed uniformly and allowed to stand at room temperature for 30 minutes, and then the reaction progress was detected with an Envision plate reader (Perkin Elmer) at wavelengths of 620 nm and 665 nm. The ratio of 665/620 is positively correlated with the phosphorylation degree of the substrate, thereby detecting the activity of VEGFR2 kinase.
Affinity data for this assay
 
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