| Assay Method Information | |
| | Time-Resolved Fluorescence Resonance Energy Transfer (TR-FRET) |
| Description: | Using human recombinant KRAS G12D, SOS and c-Raf proteins, the inhibitory activity of subject compounds on the complex formation of the proteins was examined by a time-resolved fluorescence resonance energy transfer (TR-FRET) method.Biotinylated AviTag-KRAS G12D (amino acid region of 1-185, GDP) (2.5 μL; 400 nM) and subject compounds dissolved in an assay buffer (50 mM HEPES, 150 mM NaCl, 5 mM MgCl2, 0.05% Tween 20, pH 7.0) were added to a 384-well plate (from Corning) in a liquid volume of 2.5 μL at 40,000 nM to 40 nM. Son of Sevenless (SOS) (amino acid region of 564-1049, 2.5 μL; 1.3 μM) and c-Raf (amino acid region of 51-131) GST (2.5 μL; 130 nM) containing GTP (from Sigma-Aldrich; 2 μM) were added to the plate, and the plate was allowed to stand for 1 hour at room temperature. Then, a mixture liquid (10 μL) of LANCE Ulight-anti-GST (from PerkinElmer; 120 nM) and LANCE Eu-W1024 labeled Streptoavidin (from PerkinElmer; 100 ng/mL) was added, and the 620-nm and 665-nm fluorescence intensities were measured using EnVision 2104 (from PerkinElmer) under the conditions of an excitation wavelength of 337 nm. After standardizing the values with the fluorescence intensity at a reference wavelength of 620 nm, the 50% inhibitory concentrations (IC50) were calculated by Sigmoid-Emax model nonlinear regression analysis with the signaling value in treatment with the solvent taken as 0% inhibition and with the signaling value without the addition of GTP taken as 100% inhibition. |
| Affinity data for this assay | |
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