S200 column

Manufactured by Cytiva

The S200 column is a size-exclusion chromatography column designed for the purification and analysis of macromolecules such as proteins, peptides, and other biomolecules. The column features a resin-based packing material that allows for the separation of molecules based on their size and molecular weight.

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Lab products found in correlation

Mini protean tgx stain free gel, by Bio-Rad (1 mentions) Laemmli buffer, by Bio-Rad (1 mentions) Bira500 kit, by Avidity (1 mentions) Streptrap hp column, by Cytiva (1 mentions)

2 protocols using s200 column

Biotinylation of Trimeric Hemagglutinins

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Biotinylation of the HAs was performed using the BirA500 kit (Avidity) following the manufacturer’s instructions. To compensate for the reduced activity in PBS, twice the amount of BirA was added and the reaction was additionally supplied with twice the amount of biotin using the supplied BIO-200. The biotinylation reaction was allowed to proceed for 1.5 hr at 30°C before 0.2 µm filtering and purification over an S200 column (Cytiva). The trimeric HAs were then concentrated and flash-frozen in liquid nitrogen for single-use aliquots. Biotinylated HAs were quality controlled by a gel shift assay. Approximately 2 µg of biotinylated HA was heated in non-reducing Laemmli buffer (Bio-Rad, #1610737) at 95°C for 5 min. Once cooled to room temperature, excess streptavidin was added and allowed to incubate for at least 5 min. As a control, samples were run with PBS added rather than streptavidin. The mixture was then run on a Mini-PROTEAN TGX Stain-Free gel (Bio-Rad, #4568096) and imaged. All biotinylated HAs shifted in the presence of streptavidin, indicating successful biotinylation.

Phillips A.M., Maurer D.P., Brooks C., Dupic T., Schmidt A.G, & Desai M.M. (2023). Hierarchical sequence-affinity landscapes shape the evolution of breadth in an anti-influenza receptor binding site antibody. eLife, 12, e83628.

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CCHFV Gn/Gc Fusion Protein Expression

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A construct containing CCHFV strain IbAr10200 GPC residues 1 to 1579 with an internal deletion of residues 691-1040 replaced with a 29-amino acid glycine/serine linker, fusing Gn to Gc was expressed in stably transfected Schneider 2 (S2) insect cells. The N-terminal MLD (GPC residues 1 to 247) was cleaved at the native furin cleavage site (GPC residues 244 to 247), and GP38 was also cleaved via an introduced furin cleavage site, replacing the native SKI-1 site (residues 516 to 519). The resulting Gn/Gc fusion protein was secreted and harvested from clarified S2 culture supernatant via a C-terminal double strep-tag II sequence using affinity chromatography (5 ml. StrepTrap™ HP column, Cytiva) using phosphate buffered saline (PBS) supplemented with 150 mM NaCl and eluted using the same buffer with 2.5 mM desthiobiotin. The Gn/Gc protein was then purified by size-exclusion chromatography (SEC) using an S200 column (Cytiva) in PBS supplemented with 150 mM NaCl.

Fels J.M., Maurer D.P., Herbert A.S., Wirchnianski A.S., Vergnolle O., Cross R.W., Abelson D.M., Moyer C.L., Mishra A.K., Aguilan J.T., Kuehne A.I., Pauli N.T., Bakken R.R., Nyakatura E.K., Hellert J., Quevedo G., Lobel L., Balinandi S., Lutwama J.J., Zeitlin L., Geisbert T.W., Rey F.A., Sidoli S., McLellan J.S., Lai J.R., Bornholdt Z.A., Dye J.M., Walker L.M, & Chandran K. (2021). Protective neutralizing antibodies from human survivors of Crimean-Congo hemorrhagic fever. Cell, 184(13), 3486-3501.e21.

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