Amylose resin

The pET-26-MBP-ELIC was provided by Raimund Dutzler (Addgene plasmid # 39239) and was used for the generation of both WT and mutant ELIC. Site-directed mutagenesis was conducted using the Quikchange method and subsequently confirmed by Sanger sequencing (Genewiz). WT and mutant ELIC were expressed as previously reported^{15 (link),54 (link)} in OverExpress C43 (DE3) E. Coli (Lucigen). Terrific Broth (Sigma) was used to grow cultures which were induced with 0.1 mM IPTG for ~16 h at 18 °C. The cells were pelleted, resuspended in Buffer A (20 mM Tris pH 7.5, 100 mM NaCl) with complete EDTA-free protease inhibitor (Roche), and lysed using an Avestin C5 emulsifier at ~15,000 psi. Membranes were pelleted by ultracentrifugation, resuspended in Buffer A, solubilized with 1% DDM (Anatrace), and incubated with amylose resin (New England Biolabs) for 2 h. The amylose resin was washed with 20 bed volumes of Buffer A containing 0.02% DDM and 0.05 mM TCEP (ThermoFisher Scientific), and eluted with 40 mM maltose (Sigma). The eluted protein was digested with HRV-3C protease (ThermoFisher Scientific) (10 units per mg ELIC) overnight at 4 °C then purified over a Sephadex 200 Increase 10/300 (GE Healthcare Life Sciences) size exclusion column in Buffer A with 0.02% DDM.

All proteins were added in 1:1 stoichiometric ratio and incubated together in binding buffer (50 mM HEPES pH 7.4, 150 mM NaCl, 5% glycerol, and 0.125 mg/mL BSA) with 20 µL of Glutathione Sepharose 4b resin (Cytiva), amylose resin (NEB), or Pierce streptavidin magnetic beads (Thermo Scientific). After extensive washing (50 mM HEPES pH 7.4, 500 mM NaCl, 5% glycerol, and 0.125 mg/mL BSA), 30 µL 4× loading dye was added to resin. Supernatants were loaded and protein analyzed through SDS-PAGE. After transfer to nitrocellulose membrane, GST-HRAS/-KRAS was probed with GST antibody (Santa Cruz Biotechnologies SC-138), KRAS with RAS antibody (Cell Signaling 67648S), and BRAF NTs/ BRAF-KD with His antibody (Sigma SAB5600227). Finally, western blots were imaged on Cytiva Typhoon imager.

All proteins were added in 1:1 stoichiometric ratio and incubated together in binding buffer (50 mM HEPES pH 7.4, 150 mM NaCl, 5% Glycerol, and 0.125 mg/mL BSA) with 20 μL of glutathione sepharose 4b resin (Cytiva), amylose resin (NEB), or Pierce streptavidin magnetic beads (ThermoScientific). After extensive washing (50 mM HEPES pH 7.4, 500 mM NaCl, 5% Glycerol, and 0.125 mg/mL BSA), 30 μL 4x loading dye was added to resin. Supernatant were loaded and protein analyzed through SDS-PAGE. After transfer to nitrocellulose membrane, GST-HRAS/-KRAS was probed with GST antibody (Santa Cruz Biotechnologies SC-138), KRAS with RAS antibody (Cell Signaling 67648S) and BRAF NTs/ BRAF KD with His antibody (Sigma SAB5600227). Finally, western blots were imaged on Cytiva Typhoon imager.