Superose 6 increase 10 300 column

Manufactured by GE Healthcare

Sourced in Sweden

The Superose 6 Increase 10/300 column is a size exclusion chromatography column designed for the separation and purification of biomolecules such as proteins, peptides, and nucleic acids. It has a separation range of 5,000 to 5,000,000 Da and can be used with aqueous or organic solvents.

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Lab products found in correlation

Astra 6, by Wyatt Technology (3 mentions) Histrap hp, by GE Healthcare (3 mentions) Bio beads sm 2, by Bio-Rad (3 mentions) Freestyle 293 f cells, by Thermo Fisher Scientific (3 mentions) Hitrap, by Cytiva (3 mentions) Minidawn treos, by Wyatt Technology (2 mentions) Dawn heleos 2 system, by Wyatt Technology (2 mentions) Optilab t rex ri detector, by Wyatt Technology (2 mentions) 100 kda cut off centrifugal filters, by Merck Group (2 mentions) Dawn 8 mals system, by GE Healthcare (2 mentions) Superdex 200 increase 10 300 column, by GE Healthcare (2 mentions) Kta purifier 10 system, by GE Healthcare (2 mentions) Amicon concentrator, by Merck Group (2 mentions) Protease inhibitor mix, by Serva Electrophoresis (2 mentions) 1260 bioinert hplc, by Agilent Technologies (2 mentions) Kta pure system, by GE Healthcare (2 mentions) Hitrap q column, by GE Healthcare (2 mentions) Low molecular weight gel filtration calibration kit, by GE Healthcare (1 mentions) Akta pure, by GE Healthcare (1 mentions) Complete edta free protease inhibitor, by Merck Group (1 mentions)

Close spelling variants of this product

Superose 6 column (200 citations) Superose 6 Increase 10/300 GL column (191 citations) Superose 6 (82 citations) Superose 6 10/300 column (79 citations) Superose 6 Increase column (51 citations) Superose 6 10/300 (36 citations) Superose 6 Increase (24 citations) Superose 6 Increase 10/300 (18 citations) Superose 6 increase 10/300 GL size exclusion column (11 citations) Superose 6 increase 10/300 GL size exclusion chromatography column (5 citations)

67 protocols using superose 6 increase 10 300 column

Oligomeric Status Determination of Fusion Proteins

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To determine the oligomeric status of the purified fusion proteins, size exclusion chromatography (SEC) was performed in non-denaturing conditions. 500 µL of the proteins ranging from 2 mg/mL – 4 mg/mL concentration were loaded on an analytical Superose 6 Increase 10/300 column (GE Healthcare) and eluted in 1x PBS (pH 7.4) at 0.4 mL/min flow rate on an Äkta pure chromatography system. A low molecular weight gel filtration calibration kit (product code-28403841) (GE Healthcare) was used for calibrating the column. For SEC-MALS (SEC-Multiangle light scattering), proteins were separated on a Superose 6 Increase 10/300 column (GE Healthcare) in 1x PBS (pH 7.4) at a flow rate of 0.4 mL/min. SEC resolved peaks were subjected to an in-line MALS detector (mini-DAWN TREOS, Wyatt Technology corp.) and a refractive index monitor (WATERS corp.) for molecular weight estimation. The data was analyzed through ASTRA 6.0 software (Wyatt Technology), as described previously (31 (link)).

Kar U., Khaleeq S., Garg P., Bhat M., Reddy P., Vignesh V.S., Upadhyaya A., Das M., Chakshusmathi G., Pandey S., Dutta S, & Varadarajan R. (2022). Comparative Immunogenicity of Bacterially Expressed Soluble Trimers and Nanoparticle Displayed Influenza Hemagglutinin Stem Immunogens. Frontiers in Immunology, 13, 890622.

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SEC-MALS Analysis of Ska Complex

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SEC-MALS was performed on a Dawn Heleos II System with an Optilab T-rEX RI detector (Wyatt) and a 1260 Inifinity II LC system (Agilent). The Superose 6 increase 10/300 column (GE Healthcare) was pre-equilibrated with running buffer (50 mM HEPES pH 8.0, 200 mM NaCl, 10% Glycerol and 1 mM TCEP). Analysis was performed at room temperature with 100 µl Ska complex that was pre-diluted in running buffer from 6.7 mg/ml to 1 mg/ml.

Huis in 't Veld P.J., Volkov V.A., Stender I.D., Musacchio A, & Dogterom M. (2019). Molecular determinants of the Ska-Ndc80 interaction and their influence on microtubule tracking and force-coupling. eLife, 8, e49539.

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SARS-CoV-2 Spike Protein Expression and Purification

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The SARS-CoV spike construct (Tor2 strain) for recombinant spike protein expression contains the mammalian-codon-optimized gene encoding residues 1–1190 of the spike followed by a C-terminal T4 fibritin trimerization domain, a HRV3C cleavage site, 8x-His tag and a Twin-strep tags subcloned into the eukaryotic-expression vector pαH. Residues at 968 and 969 were replaced by prolines for generating stable spike proteins as described previously [28 (link)]. The spike plasmid was transfected into FreeStyle 293F cells and cultures were harvested at 6-day post-transfection. Proteins were purified from the supernatants on His-Complete columns using a 250 mM imidazole elution buffer. The elution was buffer exchanged to Tris-NaCl buffer (25 mM Tris, 500 mM NaCl, pH 7.4) before further purification using Superose 6 increase 10/300 column (GE Healthcare). Protein fractions corresponding to the trimeric spike proteins were collected and concentrated.

Wu N.C., Yuan M., Bangaru S., Huang D., Zhu X., Lee C.C., Turner H.L., Peng L., Yang L., Burton D.R., Nemazee D., Ward A.B, & Wilson I.A. (2020). A natural mutation between SARS-CoV-2 and SARS-CoV determines neutralization by a cross-reactive antibody. PLoS Pathogens, 16(12), e1009089.

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Recombinant Omicron S Ectodomain Production

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The vector of Omicron S ectodomain with HexaPro mutations, “GSAS” substitution at furin cleavage site (residues 682-285) and a C-terminal T4 fibritin trimerization was constructed as previously reported²¹ (link) and transfected into HEK293F cells for expression.
After 72 h, the supernatants were harvested and filtered for affinity purification by Histrap HP (GE Healthcare). The protein was then loaded onto a Superose 6 increase 10/300 column (GE Healthcare) in 20 mM Tris pH 8.0, 200 mM NaCl.

Wu J., Chen Z., Gao Y., Wang Z., Wang J., Chiang B.Y., Zhou Y., Han Y., Zhan W., Xie M., Jiang W., Zhang X., Hao A., Xia A., He J., Xue S., Mayer C.T., Wu F., Wang B., Zhang L., Sun L, & Wang Q. (2023). Fortuitous somatic mutations during antibody evolution endow broad neutralization against SARS-CoV-2 Omicron variants. Cell Reports, 42(5), 112503.

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Stabilized SARS-CoV-2 Spike Protein Production

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The SARS-CoV-2 S construct used for negative-stain EM contains the mammalian-codon-optimized gene encoding residues 1-1208 of the S protein (GenBank: QHD43416.1), followed by a C-terminal T4 fibritin trimerization domain, an HRV3C cleavage site, 8x-His tag and a Twin-strep tags subcloned into the eukaryotic-expression vector pcDNA3.4. Three amino-acid mutations were introduced into the S1–S2 cleavage site (RRAR to GSAS) to prevent cleavage and two stabilizing proline mutations (K986P and V987P) to the HR1 domain. For additional S stabilization, residues T883 and V705 were mutated to cysteines to introduce a disulphide bond. The S plasmid was transfected into 293F cells and supernatant was harvested at 6 days post transfection. S protein was purified by running the supernatant through a streptactin column and then by size exclusion chromatography using a Superose 6 increase 10/300 column (GE Healthcare Biosciences). Protein fractions corresponding to the trimeric S protein were collected and concentrated.

Liu H., Wu N.C., Yuan M., Bangaru S., Torres J.L., Caniels T.G., van Schooten J., Zhu X., Lee C.C., Brouwer P.J., van Gils M.J., Sanders R.W., Ward A.B, & Wilson I.A. (2020). Cross-Neutralization of a SARS-CoV-2 Antibody to a Functionally Conserved Site Is Mediated by Avidity. Immunity, 53(6), 1272-1280.e5.

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Structural Characterization of NiV F-Antibody Complex

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Purified FLAG-tagged NiV F N100C/A119C ectodomain was combined with an excess molar ratio of 5B3 Fab and incubated on ice for 1 hour before injection on a Superose 6 Increase 10/300 column (GE Healthcare) equilibrated in a buffer containing 50 mM Tris pH 8.0 and 150 mM NaCl. The fractions containing the complex were quality-controlled by negative staining EM, pooled, buffer-exchanged and concentrated.

Dang H.V., Chan Y.P., Park Y.J., Snijder J., Da Silva S.C., Vu B., Yan L., Feng Y.R., Rockx B., Geisbert T.W., Mire C.E., Broder C.C, & Veesler D. (2019). An antibody against the F glycoprotein inhibits Nipah and Hendra virus infections. Nature Structural & Molecular Biology, 26(10), 980-987.

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Negative-stain EM Complexes Preparation

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Complexes for negative-stain EM were prepared similarly to the previously described method, except ELISA EC₅₀ was not used to guide serum Fab concentration to be used (Bianchi et al., 2018 (link)). Briefly, after buffer exchanging into TBS, up to ~1 mg of total Fab was incubated overnight with 10 μg BG505 trimers at RT in ~50 μL total volume. Complexes were then purified by SEC using Superose 6 Increase 10/300 column (GE Healthcare) in order to remove unbound Fab. The flow-through fractions containing the complexes were pooled and concentrated using 100 kDa cutoff centrifugal filters (EMD Millipore). The final trimer concentration was titrated to ~0.04 mg/mL prior to application onto carbon-coated copper grids. Of note, depending on sample availability, not all complexes were prepared using identical Fab concentrations during incubation with trimers, with samples from animals where serum material was limiting resulting in lower Fab:trimer ratio. For animal 12–046, week 10 and 26 samples were insufficient to include in the time-course study as the purified total Fab concentration was severalfold lower than the rest of the animals (see Table S1).

Nogal B., Bianchi M., Cottrell C.A., Kirchdoerfer R.N., Sewall L.M., Turner H.L., Zhao F., Sok D., Burton D.R., Hangartner L, & Ward A.B. (2020). Mapping Polyclonal Antibody Responses in Non-human Primates Vaccinated with HIV Env Trimer Subunit Vaccines. Cell reports, 30(11), 3755-3765.e7.

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PAPP-A Dimerization Mechanism Analysis

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For the SEC assay to examine dimerization mechanism, purified WT PAPP-A, PAPP-A (E483A), PAPP-A2 WT, PAPP-A monomeric mutants, and C-terminal truncation constructs (PAPP-A (1132) and PAPP-A (1267)) were thawed and centrifuged at 15,000 g for 5 min at 4 °C to remove any potential precipitates. Concentration of the PAPP-A proteins were normalized to 1.4 μM, then 0.2 mL of each protein was injected onto a Superose 6 Increase 10/300 column (Cytiva 29-0915-96) connected to an AKTA Pure (GE Healthcare). The system was run at 0.5 mL/min for 1 h using 1X PBS as the mobile phase. UV280 measurements were obtained directly from the instrument. The fractions from retention volume between 11.5-16.5 ml were run on a 4-12% Bis-Tris SDS-PAGE, stained with Coomassie Protein Stain (InstantBlue® ab119211) and destained with Milli Q Water.

Judge R.A., Sridar J., Tunyasuvunakool K., Jain R., Wang J.C., Ouch C., Xu J., Mafi A., Nile A.H., Remarcik C., Smith C.L., Ghosh C., Xu C., Stoll V., Jumper J., Singh A.H., Eaton D, & Hao Q. (2022). Structure of the PAPP-ABP5 complex reveals mechanism of substrate recognition. Nature Communications, 13, 5500.

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Purification of MBP-tagged Ssp6 protein

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For purification of MBP-Ssp6, E. coli C43 (DE3) cells transformed with a pNIFTY-MBP-derived plasmid encoding Ssp6 fused with N-terminal MBP were inoculated to a starting OD₆₀₀ 0.05 in 4 L LB, grown at 30 °C, 200 rpm for 3 h, induced with 0.5 mM IPTG and then incubated for 16 h at 16 °C. Cells were recovered by centrifugation (4000 × g, 30 min), resuspended in 40 mL of Buffer A (50 mM Tris-HCl pH 8, 500 mM NaCl) in presence of cOmplete™ EDTA-free protease inhibitor (Sigma) and lysed using an EmulsiFlex-C3 homogenizer (Avestin). The lysate was cleared by centrifugation (14,000 × g, 45 min, 4 °C), filtered through a 0.45 µm filter, and loaded onto 1 mL MBP Trap™ HP column (GE Healthcare) following equilibration with Buffer A. Elution was achieved using 10 column volumes of Buffer B (50 mM Tris-HCl pH 8, 500 mM NaCl, 10 mM maltose). The eluted fraction was separated by size exclusion chromatography using a Superose 6 Increase 10/300 column (GE Healthcare) and a buffer containing 50 mM Tris-HCl pH 8, 150 mM NaCl, 10% glycerol.

Mariano G., Trunk K., Williams D.J., Monlezun L., Strahl H., Pitt S.J, & Coulthurst S.J. (2019). A family of Type VI secretion system effector proteins that form ion-selective pores. Nature Communications, 10, 5484.

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Lectin-BG505 SOSIP.664 Binding Assay

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Lectin and BG505 SOSIP.664 were combined at varying molar ratios (using the molecular weights of BG505 SOSIP.664 trimer and lectin tetramer) of 0.75:1, 1.5:1, 3.75:1, and 7.5:1, incubated at room temperature for 15 min, and then run on a 4–16% BN-PAGE gel according to manufacturer’s recommendations. A separate Lectin and BG505 SOSIP.664 incubated sample (6:1 molar ratio) was subjected to centrifugation (14,000 g, 10 min, 4 °C) and loaded onto a Superose 6 Increase 10/300 column (GE Healthcare). The higher molecular weight peak, corresponding to the expected MW for SOSIP.664 trimer, was pooled, concentrated, and analyzed by EM.

Hopper J.T., Ambrose S., Grant O.C., Krumm S.A., Allison T.M., Degiacomi M.T., Tully M.D., Pritchard L.K., Ozorowski G., Ward A.B., Crispin M., Doores K.J., Woods R.J., Benesch J.L., Robinson C.V, & Struwe W.B. (2017). The Tetrameric Plant Lectin BanLec Neutralizes HIV through Bidentate Binding to Specific Viral Glycans. Structure (London, England : 1993), 25(5), 773-782.e5.

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