HEK-293F cells were seeded at 1 × 106 cells/mL in Freestyle 293 Expression Medium (Gibco). The next day, a transfection mix was prepared (for 200 mL of cells) of 72 mg of Wuhan-hu1 Spike-Avi-His tag plasmid and 18 mg of BirA plasmid97 (link) into 11 ml of Opti-MEM, alongside 2ml of PEI-Max, and left to incubate at 37°C 5%CO2 in a shaking incubator for 7 days before harvesting for purification. The supernatant was purified using 2 mM imidazole buffer (Sigma-Aldrich) buffer during binding to a His GraviTrap (Cytiva) column and 500 mM imidazole buffer for elution. The eluted protein was then concentrated with a 100KD Amicon Ultra concentrator (Millipore) and washed with PBS before quantification using a NanoDrop. Biotinylated protein was then further purified through size exclusion chromatography using an AKTA pure system with a Superdex 200 Increase 10/300 GL column (Sigma-Aldrich) to select for fractions containing trimeric Spike.
Superdex 200 increase 10 300 gl column
Manufactured by Merck Group
The Superdex 200 Increase 10/300 GL column is a size exclusion chromatography column designed for the separation and purification of molecules based on their size and shape. It has a separation range of 10,000 to 600,000 daltons and can be used with a variety of aqueous buffers and organic solvents. The column dimensions are 10 mm in diameter and 300 mm in length.
Automatically generated - may contain errors
Lab products found in correlation
Freestyle 293 expression medium, by Thermo Fisher Scientific (3 mentions)
His gravitrap, by Cytiva (3 mentions)
100kd amicon ultra concentrator, by Merck Group (2 mentions)
Imidazole, by Merck Group (2 mentions)
Imidazole buffer, by Merck Group (1 mentions)
Imidazole, by Cytiva (1 mentions)
Captureselect igg fc resin, by Thermo Fisher Scientific (1 mentions)
Papaya latex papain, by Merck Group (1 mentions)
Mwco concentrator, by Merck Group (1 mentions)
Dawn heleos 2, by Wyatt Technology (1 mentions)
Nanodrop, by Merck Group (1 mentions)
6 protocols using superdex 200 increase 10 300 gl column
1
Production and Purification of SARS-CoV-2 Spike Protein
2
Biotinylation of SARS-CoV-2 Spike and RBD
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To produce biotinylated spike and receptor binding domain (RBD) protein, HEK-293F cells were seeded at 1 × 106 cells/mL in Freestyle™ 293 Expression Medium (Gibco). The next day, a transfection mix was prepared (for 200 mL of cells) of 72 μg of spike-Avi-His tag or RBD-Avi-His tag plasmid and 18 μg of BirA plasmid36 (link),88 (link) into 11 mL of Opti-MEMTM, alongside 2 mL of PEI-Max® and 3 mL of 10 mM biotin, and left to incubate at 37°C 5% CO2 in a shaking incubator for 7 days before harvesting for purification. The supernatant was purified using an imidazole (Sigma-Aldrich) buffer at a final concentration of 20 mM during binding to the His GraviTrap™ (Cytiva) column and 500 mM imidazole for elution. The eluted protein was then concentrated with a 100KD Amicon® Ultra concentrator (Millipore) and washed with PBS before quantification using a NanoDropTM. Biotinylated protein was then further purified through size exclusion chromatography using an AKTA™ pure system with a Superdex® 200 Increase 10/300 GL column (Sigma-Aldrich) to select for fractions containing trimeric spike or RBD protein.
3
Protein Fractionation via Gel Filtration
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The extracted protein mixtures were filtered through a 0.2-μm filter, then loaded onto a Superdex 200 Increase 10/300 GL column (Sigma-Aldrich). A buffer containing 50 mM Tris–HCl (pH 7.5), 150 mM NaCl, 10 mM MgCl2, 2.5 mM EDTA, 1 mM DTT, 10% (v/v) glycerol, 0.1% (v/v) NP-40, and 1 mM PMSF was used for column equilibration. The proteins were fractionated at a flow rate of 0.5 ml/min and collected in 0.5-ml aliquots. The gel-filtration assay was performed at the Korea Basic Science Institute in Ochang, Korea.
4
Papain Digestion of Purified IgG
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Fabs of 19.7E were generated by papain digestion of purified IgG. First, a buffered aqueous suspension of papaya latex papain (Sigma Aldrich) was activated by incubating in 100 mM Tris, 2 mM EDTA, 10 mM L-cysteine at 37°C for 15 mins. Next, IgG was incubated with activated papain in 100 mM Tris, 2 mM EDTA, 10 mM L-cysteine at a ratio of 40 μg activated papain per 1 mg of purified IgG for 5 hours at 37°C. The reaction was quenched by adding iodoacetamide to a final concentration of 0.03 M. Undigested IgG and Fc fragments were removed by a 2 h incubation with CaptureSelect IgG-Fc resin (Thermo Fisher Scientific). Resin was spun down and the supernatant run on a Superdex 200 increase 10/300 GL column (Sigma-Aldrich) size exclusion column using TBS as its running buffer. Fractions from 15.5-16.5 mL elution volume were collected and concentrated in a MWCO concentrator (Millipore) with a 10 kDa cutoff.
5
SEC-MALS Analysis of Protein Oligomerization
6
Biotinylated SARS-CoV-2 Spike and RBD Protein Production
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To produce biotinylated spike and receptor binding domain (RBD) protein, HEK-293F cells were seeded at 1×106 cells/ml in Freestyle™ 293 Expression Medium (Gibco). The next day, a transfection mix was prepared (for 200ml of cells) of 72μg of spike-Avi-His tag or RBD-Avi-His tag plasmid and 18μg of BirA plasmid (Graham et al., 2021 (link); Seow et al., 2020 ) into 11ml of Opti-MEM™, alongside 2ml of PEI-Max® and 3ml of 10mM biotin, and left to incubate at 37°C 5% CO2 in a shaking incubator for 7 days before harvesting for purification. The supernatant was purified using an imidazole (Sigma-Aldrich) buffer at a final concentration of 20mM during binding to the His GraviTrap™ (Cytiva) column and 500mM imidazole for elution. The eluted protein was then concentrated with a 100KD Amicon® Ultra concentrator (Merck) and washed with PBS before quantification using a NanoDrop™. Biotinylated protein was then further purified through size exclusion chromatography using an AKTA™ pure system with a Superdex® 200 Increase 10/300 GL column (Sigma-Aldrich) to select for fractions containing trimeric spike or RBD protein.
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