| Assay Method Information | |
| | TR-FRET Assay |
| Description: | The following binding partners have been used in this assay. Biotinylated KRAS G12D protein corresponding to KRAS (amino acids 1-169, with the following changes to the natural protein: G12D) was expressed in E. coli with a carboxy-terminal Avi tag (amino acid sequence GGGLNDIFEAQKIEWHE). GST-tagged SOS1 protein corresponding to SOS1 (amino acids 564-1049) with an amino-terminal GST-tag and a Tobacco-etch-virus (TEV) protease cleavage site was expressed in E. coli and purified by affinity chromatography on a GSH-column, followed by desalting (HiPrep 26/10 Desalting, GE Healthcare) into 20 mM TRIS, 200 mM NaCl, 10% Glycerol, 1 mM DTT, pH 8.0. The tag was not cleaved.Compounds are dispensed onto assay plates (Proxiplate 384 PLUS, white, PerkinElmer) using an Access Labcyte Workstation with the Labcyte Echo 55× from a DMSO solution. For the chosen highest assay concentration of 500 μM or 100 μM (this can be changed upon request), 150 nl of compound solution are transferred from a 50 mM or 10 mM DMSO compound stock solution. Compounds are tested in duplicates. A series of 11 concentrations is transferred for each compound at which each concentration is fivefold lower than the previous one. DMSO is added such that every well has a total of 150 nl compound solution. The assay runs on a fully automated robotic system. For the assay 15 μl containing KRAS G12D protein (15 nM final assay concentration), SOS1 (10 nM final assay concentration), GTP (10 μM final assay concentration), Lance Eu-W1024 labeled Streptavidin (1.5 nM final assay concentration) and Anti-GST surelight APC (30 nM final assay concentration) mixed in assay buffer (1×PBS; 0.05% Tween20; 0.1% BSA; filtered) are added to the 150 nl of compounds. Plates are kept at room temperature. After 60 minutes incubation time the TR-FRET signal is measured in a PerkinElmer Envision HTS Multilabel Reader using the TR-FRET LANCE Ultra specs of PerkinElmer. |
| Affinity data for this assay | |
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