| Assay Method Information | |
| | KRAS::SOS1 AlphaScreen Binding Assay |
| Description: | This assay is used to examine the potency with which compounds inhibit the protein protein interaction between SOS1 and KRAS G12D in a defined biochemical setting. Low IC50 values of given compounds are indicative of high potency of the SOS1 inhibitor compounds in this assay setting.Reagents: GST-TEV-SOS1 (564-1049) and His-TEV-Avi-KRAS G12D (1-169) are purchased from Viva Biotech (Shanghai) Ltd. GDP (Sigma, Cat. G7127) AlphaLISA Glutathione Acceptor Beads (PerkinElmer, Cat. AL109C) AlphaScreen Streptavidin Donor Beads (PerkinElmer, Cat. 6760002S) Assay plates: ProxiPlate-384 Plus, White 384-shallow well Microplate (PerkinElmer, Cat. 6008280)Assay Buffer: PBS, pH 7.4 (Gibco, Cat. 10010023) 0.05% Tween 20 (Sigma, Cat. P7949 100 ML) 0.1% Bovine Serum Albumin (BSA) (Sigma, Cat. A1933 5 G)Assay Protocol:SOS1 inhibitor compounds are diluted to a final start concentration of 1 PM. Serial dilutions of compounds are made using Tecan D300e Digital Dispenser in 9 concentrations with serial 1:3 dilutions. 100 nL of compound solution is transferred to the 384-well assay plate per well, covering a range between 1 μM and 0.15 nM minimum in duplicate. 10 nM (final assay concentration) KRAS G12D, 5 nM (final assay concentration) SOS1 and 10 μM (final assay concentration) GDP are mixed in assay buffer, and 5 μL of KRAS SOS1 GDP mix is added into the assay plate to the 100 nL of compound solution (final dilution in the assay 1:100, final DMSO concentration 1%). After a 30 min incubation, AlphaLISA Glutathione Acceptor Beads and AlphaScreen Streptavidin Donor Beads are mixed in assay buffer at a concentration of 5 g/mL (final assay concentration), and 5 μL of bead mix is added into the assay plate. Plates are kept at room temperature in a darkened incubator for 3 h. After a 3 h incubation, the signal is determined using Envision (PerkinElmer). The excitation wavelength is 680 nm, and emission 615 nm. IC50 values are calculated and analyzed using GraphPad Prism. |
| Affinity data for this assay | |
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