| Assay Method Information | |
| | KRAS-GDP Protein Assay |
| Description: | 6> Add 5 μL of KRAS solution prepared with Buffer 1 to each well of the high-signal control group and compound test wells. Add 5 μL of Buffer 1 to each well of the low-signal control group. The final concentration of KRAS in the reaction is 5 nM (initially 40 nM, optimized to 5 nM). Incubate at room temperature for 1 hour.7> Add 5 μL of a solution containing SOS1 and GTP prepared with Buffer 2 to each well. Final concentrations in the reaction: 50 nM for SOS1 (initially 250 nM) and 125 μM for GTP (initially 625 μM). Incubate at room temperature for 1 hour.8> Add 5 μL of a solution containing cRAF and AlphaLISA Nickel Acceptor beads prepared with Buffer 2 to each well. Final concentrations in the reaction: 50 nM for cRAF and 20 μg/mL for AlphaLISA Nickel Acceptor beads. Incubate at room temperature for 1 hour.9> Add 5 μL of an AlphaScreen GSH Donor beads solution prepared with Buffer 2 to each well. Final concentration in the reaction: 20 μg/mL. Incubate at room temperature for 1 hour.10> Measure the signal values using an EnVision plate reader. Excitation wavelength: 680 nm, Emission wavelength: 615 nm. |
| Affinity data for this assay | |
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