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Amylose resin

Manufactured by Thermo Fisher Scientific

Amylose resin is a chromatography medium used for the purification of recombinant proteins and other biomolecules. It functions by selectively binding to proteins that have an affinity for amylose, allowing for their isolation and separation from other components in a sample.

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4 protocols using amylose resin

1

Heterologous Expression and Purification of ELIC

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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 reported15 (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.
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2

Affinity Capture of HRAS, KRAS, and BRAF Proteins

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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.
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3

Protein-Protein Interaction Assay

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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.
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4

Protein-Protein Interaction Assay

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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.
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