Superdex 75 10 300 gl gel filtration column

Manufactured by GE Healthcare

Sourced in United Kingdom, Sweden

The Superdex 75 10/300 GL gel filtration column is a prepacked size exclusion chromatography column designed for the separation and purification of proteins, peptides, and other biomolecules. The column is filled with a highly cross-linked agarose-dextran matrix that provides efficient and reproducible separations based on the molecular size of the analytes.

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Lab products found in correlation

Histrap nickel column, by GE Healthcare (5 mentions) Freestyle 293 expression medium, by Thermo Fisher Scientific (5 mentions) Expi293f cells, by Thermo Fisher Scientific (5 mentions) Strep tactin xt resin, by IBA Lifesciences (2 mentions) Pdisplay bira er, by Addgene (2 mentions) D biotin, by Merck Group (2 mentions) Akta fplc system, by GE Healthcare (1 mentions) J 815 cd spectrometer, by Jasco (1 mentions) Hitrap q hp column, by GE Healthcare (1 mentions) Ktaprime plus, by GE Healthcare (1 mentions) Amicon ultrafiltration device, by Merck Group (1 mentions) Dawn heleos 2 8, by Wyatt Technology (1 mentions) Superdex 200 10 300 gl column, by GE Healthcare (1 mentions) Astra 6, by Wyatt Technology (1 mentions) Optilab t rex, by Wyatt Technology (1 mentions)

Close spelling variants of this product

Superdex 75 (385 citations) Superdex 75 10/300 GL column (288 citations) Superdex 75 column (263 citations) Superdex 75 10/300 GL (133 citations) Superdex 75 10/300 column (83 citations) Superdex 75 10/300 (52 citations) Superdex 75 gel filtration column (33 citations) Superdex™ 75 GL column (5 citations) Superdex 75 10/300 GL gel (3 citations) Superdex 75 10/300 gel (3 citations)

13 protocols using superdex 75 10 300 gl gel filtration column

Rab8a Complex Formation Analysis

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The bMERB:GppNHp Rab8a_1-176 complex formation was analyzed by aSEC. 110 µM of bMERB domain and 121 µM of GppNHp Rab8_1-176 protein (Effector: Rab stoichiometry of 1:1.1) was mixed in a buffer containing 20 mM Hepes 7.5, 50 mM NaCl, 1 mM MgCl₂ and 2 mM DTE (Rab buffer) and centrifuged for 15 min at 15,700 g at 4 °C. 40 µl of the mixture was injected into a Superdex 75 10/300 GL gel filtration column (GE Healthcare) pre-equilibrated with the Rab buffer with a flow rate of 0.5 ml/min at room temperature and absorption at 280 nm was recorded.
Similarly, the complex formation between the bMERB (110 µM) domain and the CH (121 µM) domain was analyzed in the buffer containing 20 mM Hepes 7.5, 150 mM NaCl, and 2 mM DTE (CH buffer) and centrifuged for 15 min at 15700 g at 4 °C. 40 µl of the mixture was injected onto a Superdex 75 10/300 GL gel filtration column (GE Healthcare) pre-equilibrated with the CH buffer with a flow rate of 0.5 ml/min at room temperature and absorption at 280 nm was recorded.

Rai A., Bleimling N., Vetter I.R, & Goody R.S. (2020). The mechanism of activation of the actin binding protein EHBP1 by Rab8 family members. Nature Communications, 11, 4187.

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Purification of Omicron and BA RBDs

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Protein expression and purification were conducted largely as described previously (Dejnirattisai et al., 2021a (link); Zhou et al., 2021 (link)). Twin-strep tagged Omicron spike was transiently expressed in HEK293T cells and purified with Strep-Tactin XT resin (IBA lifesciences). Plasmids encoding BA.1 RBD (319–541), BA.1 RBD (330–532) and BA.2 RBD (330–532) were transiently expressed in Expi293F™ Cells (ThermoFisher), cultured in FreeStyle™ 293 Expression Medium (ThermoFisher) at 30°C with 8% CO2 for 4 days. BA.1 RBD (330–532) was expressed in the presence of 1 μg/mL kifunensine. The harvested medium was concentrated using a QuixStand benchtop system. His-tagged ACE2 and RBDs were purified with a 5 mL HisTrap nickel column (GE Healthcare), followed by a Superdex 75 10/300 GL gel filtration column (GE Healthcare).

Nutalai R., Zhou D., Tuekprakhon A., Ginn H.M., Supasa P., Liu C., Huo J., Mentzer A.J., Duyvesteyn H.M., Dijokaite-Guraliuc A., Skelly D., Ritter T.G., Amini A., Bibi S., Adele S., Johnson S.A., Constantinides B., Webster H., Temperton N., Klenerman P., Barnes E., Dunachie S.J., Crook D., Pollard A.J., Lambe T., Goulder P., Paterson N.G., Williams M.A., Hall D.R., Mongkolsapaya J., Fry E.E., Dejnirattisai W., Ren J., Stuart D.I, & Screaton G.R. (2022). Potent cross-reactive antibodies following Omicron breakthrough in vaccinees. Cell, 185(12), 2116-2131.e18.

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Calcium-Induced Conformational Dynamics of CabA

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CD spectroscopy was used to evaluate the effect of calcium on the CabA conformation. The calcium-free CabA (0.05 mg/ml) in 300 mM NaCl and 50 mM Tris-HCl, pH 8.0 was subjected to far-UV CD measurements at 25°C using a 1-mm path length quartz cuvette in a Jasco J-815 CD spectrometer (Jasco, Tokyo, Japan). The CabA protein was incubated with 10 mM CaCl₂ for 1 h at room temperature, and its CD spectra were measured under the same conditions. CD Spectra were acquired over the wavelength range of 200–260 nm and converted into mean residue ellipticity (MRE, degree cm²/dmol). Blank spectra of the buffer without the protein were subtracted.
Calcium-induced conformational change of CabA was further assessed by size-exclusion chromatography using a Superdex 75 10/300 GL gel filtration column (GE Healthcare, Waukesha, WI) installed on an AKTA FPLC system (GE Healthcare). The multimeric CabA obtained with 100 mM CaCl₂ was serially diluted to various calcium concentrations. The diluted samples were then injected onto the pre-equilibrated column with 300 mM NaCl, 20 mM Tris-HCl, pH 8.0 and corresponding concentrations of calcium and eluted at a flow rate of 0.4 ml/min at room temperature. The effluent was monitored by measuring absorbance at 280 nm. Bovine serum albumin (66 kDa) and carbonic anhydrase (29 kDa) were used as the molecular weight standards.

Park J.H., Jo Y., Jang S.Y., Kwon H., Irie Y., Parsek M.R., Kim M.H, & Choi S.H. (2015). The cabABC Operon Essential for Biofilm and Rugose Colony Development in Vibrio vulnificus. PLoS Pathogens, 11(9), e1005192.

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Purification of S100A4 Variants and NMIIA Fragment

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The S100A4 variants and NMIIA fragment were obtained as described in our recently published work [8] (link). Briefly, the His-tagged human S100A4 variants were expressed in E. coli BL21 strain, and purified by Ni²⁺-affinity chromatography. After cleavage of the His₆-tag with Tobacco Etch Virus protease, the sample were applied to phenyl-Sepharose column, washed thoroughly and eluted with EGTA containing buffer. The purified S100A4 proteins were dialysed against 20 mM Hepes pH 7.5, 20 mM NaCl, 0.1 mM TCEP buffer, concentrated and pooled at −70°C. Prior to SAXS measurements, the S100A4 alone or in complex with MPT peptide were applied to Superdex 75 10/300 GL gel filtration column (GE Healthcare, Little Chalfont, UK) and the peak corresponding to ∼24 kDa molecular weight was collected. The NMIIA fragment MPT (Tyr-Arg1894-Lys1937) was expressed with an N-terminal His₁₀-ubiquitin fusion in E. coli BL21 strain, and purified by Ni²⁺-affinity chromatography. The His-tagged ubiquitin was removed by Yeast Ubiquitin Hydrolase, while it was dialysed against buffer containing 20 mM TRIS (pH 8.0), 100 mM NaCl and 0.2 mM DTT. The completely digested sample was applied to Ni²⁺-affinity column. The peptide was in the flow-through, which was finally purified by reverse-phase HPLC on a Jupiter 300 C18 column (Phenomenex, Torrance, CA).

Duelli A., Kiss B., Lundholm I., Bodor A., Petoukhov M.V., Svergun D.I., Nyitray L, & Katona G. (2014). The C-Terminal Random Coil Region Tunes the Ca2+-Binding Affinity of S100A4 through Conformational Activation. PLoS ONE, 9(5), e97654.

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Purification of Sunflower Allergen Hel a 6

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All the chromatographic procedures were performed using an ÄKTAprime plus (GE Healthcare Life Sciences) FPLC system^{14 (link)}. The crude pollen extract (5 mg/ml) was fractionated in HiTrap Q HP column (GE Healthcare Life Sciences) equilibrated with the same buffer followed by elution of column-bound proteins using a linear gradient of 0.1–1 M NaCl. Eluted fractions containing the desired protein at a higher level of purity were pooled, dialyzed against 20 mM phosphate buffer (pH 7.4), and then concentrated in < 10 kDa cut-off Amicon Ultrafiltration device (Millipore). About 2 mg of concentrated protein was loaded onto a Superdex 75 10/300 GL gel filtration column (GE Healthcare Life Sciences)^{15 (link)}. Eluted fractions were screened by IgE-western blot using serum of 4 sunflower-sensitized patients to check for the presence of Hel a 6. The fractions containing Hel a 6 at > 90% purity were pooled, dialyzed against 1 M ammonium bicarbonate buffer (pH 8.5), and lyophilized.

Ghosh N., Sircar G., Asam C., Wolf M., Hauser M., Saha S., Ferreira F, & Bhattacharya S.G. (2020). Purification and biochemical characterization of Hel a 6, a cross-reactive pectate lyase allergen from Sunflower (Helianthus annuus L.) pollen. Scientific Reports, 10, 20177.

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SAXS Analysis of hAIPL1 Variants

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SAXS data on hAIPL1, hAIPL1_1–316, and the P376S mutants were collected using the in-line FPLC purification system at the Advance Photon Source (18 ID-BioCAT, beamline) at the Argonne National Laboratories. Concentrated protein samples of hAIPL1 (15 mg/mL), P376S (15 mg/mL), hAIPL1_1–316 (10 mg/mL) were loaded on a Superdex 75 10/300 GL gel filtration column (GE Healthcare) equilibrated against buffer B. Data were collected on the fractions as they eluted off the Superdex 75 column. The fractions before the void volume were used for buffer substraction. The SAXS data on the P351∆12 mutant were collected at the Advanced Light Source (12.3.1 SIBYLS beamline) at the Lawrence Berkeley National Laboratories. Three different concentrations of the protein (1.9, 3.8, 5.8 mg/mL) were exposed to the synchrotron radiation for 0.5, 1, 2, 4 s each. Scattering from the buffer was subtracted, and the sample scattering data were analyzed by Primus/QT (33). AUTORG and DATGNOM were used to calculate the R_g (radius of gyration) and D_max (maximum particle dimension) from a pairwise distribution function) in Primus/QT. The Kratky and P(r) plots in Fig. 3(d) and (e) for the P351∆12 mutant are scaled by factor of 0.1 for visualization purposes. This was done to bring them to a comparable scale with the other samples diluted during the in-line FPLC SAXS data collection.

Yadav R.P., Majumder A., Gakhar L, & Artemyev N.O. (2015). Extended conformation of the proline-rich domain of human aryl hydrocarbon receptor-interacting protein-like 1: implications for retina disease. Journal of neurochemistry, 135(1), 165-175.

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Recombinant SARS-CoV-2 RBD Production

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Plasmids encoding RBDs were transfected into Expi293F Cells (ThermoFisher) by PEI, cultured in FreeStyle 293 Expression Medium (ThermoFisher) at 37°C for 1 day followed by 30°C for 3 days with 8% CO₂. To express biotinylated RBDs, the RBD-BAP plasmid was co-transfected with pDisplay-BirA-ER (Addgene plasmid 20,856; coding for an ER-localized biotin ligase), in the presence of 0.8 mM D-biotin (Sigma-Aldrich). The conditioned medium was diluted 1:2 into binding buffer (50 mM sodium phosphate, 500 mM sodium chloride, pH 8.0). RBDs were purified with a 5 mL HisTrap nickel column (GE Healthcare) through His-tag binding, followed by a Superdex 75 10/300 GL gel filtration column (GE Healthcare) in 10 mM HEPES and 150 mM sodium chloride.

Huo J., Dijokaite-Guraliuc A., Liu C., Zhou D., Ginn H.M., Das R., Supasa P., Selvaraj M., Nutalai R., Tuekprakhon A., Duyvesteyn H.M., Mentzer A.J., Skelly D., Ritter T.G., Amini A., Bibi S., Adele S., Johnson S.A., Paterson N.G., Williams M.A., Hall D.R., Plowright M., Newman T.A., Hornsby H., de Silva T.I., Temperton N., Klenerman P., Barnes E., Dunachie S.J., Pollard A.J., Lambe T., Goulder P., Fry E.E., Mongkolsapaya J., Ren J., Stuart D.I, & Screaton G.R. (2022). A delicate balance between antibody evasion and ACE2 affinity for Omicron BA.2.75. Cell Reports, 42(1), 111903.

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Recombinant SARS-CoV-2 RBD Expression

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Plasmids encoding RBDs were transfected into Expi293F™ Cells (ThermoFisher) by PEI, cultured in FreeStyle™ 293 Expression Medium (ThermoFisher) at 30 °C with 8% CO₂ for 4 days. To express biotinylated RBDs, the RBD-BAP plasmid was co-transfected with pDisplay-BirA-ER (Addgene plasmid 20856; coding for an ER-localized biotin ligase), in the presence of 0.8 mM D-biotin (Sigma-Aldrich). The conditioned medium was diluted 1:2 into binding buffer (50 mM sodium phosphate, 500 mM sodium chloride, pH 8.0). RBDs were purified with a 5 mL HisTrap nickel column (GE Healthcare) through His-tag binding, followed by a Superdex 75 10/300 GL gel filtration column (GE Healthcare) in 10 mM HEPES and 150 mM sodium chloride.

Tuekprakhon A., Nutalai R., Dijokaite-Guraliuc A., Zhou D., Ginn H.M., Selvaraj M., Liu C., Mentzer A.J., Supasa P., Duyvesteyn H.M., Das R., Skelly D., Ritter T.G., Amini A., Bibi S., Adele S., Johnson S.A., Constantinides B., Webster H., Temperton N., Klenerman P., Barnes E., Dunachie S.J., Crook D., Pollard A.J., Lambe T., Goulder P., Paterson N.G., Williams M.A., Hall D.R., Fry E.E., Huo J., Mongkolsapaya J., Ren J., Stuart D.I, & Screaton G.R. (2022). Antibody escape of SARS-CoV-2 Omicron BA.4 and BA.5 from vaccine and BA.1 serum. Cell, 185(14), 2422-2433.e13.

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Yeast Two-Hybrid and SEC-MALS Analysis of NatA and NatE

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The Saccharomyces cerevisiae strain PJ69-4A (James et al., 1996) was co-transformed with pG4BDN22::ScNAA15 and pG4ADC111::ScNAA50 or pG4BDN22::AtNAA15 and pG4ADC111::AtNAA50. Double transformants were selected on synthetic complete dextrose (SDC) medium lacking leucine and tryptophan (SDC -Leu -Trp). For each transformation, four colonies were pooled and diluted to an OD 600 of 3.0 in water followed by 10-fold serial dilutions and spotted onto SDC -Leu-Trp, SDC -Leu -Trp -His and SDC -Leu -Trp -Ade. Growth was recorded after 3 days of incubation at 30 C. Transactivation controls were systematically performed for each construct.
SEC-multiangle light scattering (SEC-MALS) AtNaa50 (0.35 mg) was injected onto a Superdex 75 10/300 GL gel-filtration column (GE Healthcare) in buffer G 250 at room temperature. ScNatA (0.09 mg) and ScNatE (0.12 mg) were injected onto a Superdex 200 10/300 GL column (GE Healthcare) in buffer G 1000 at room temperature. The columns were connected to a MALS system (Dawn Heleos II 8+ and Optilab T-rEX, Wyatt Technology) for mass analysis of the eluted proteins with the Astra 6 software (Wyatt Technology).

Weidenhausen J., Kopp J., Armbruster L., Wirtz M., Lapouge K, & Sinning I. (2021). Structural and functional characterization of the N-terminal acetyltransferase Naa50. Structure (London, England : 1993), 29(5).

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SARS-CoV-2 Spike Protein Expression and Purification

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Protein expression and purification were largely the same as described previously^{7 (link),28 (link)}. Twin-strep tagged BA.4/5 and BA.4+all spikes were transiently expressed in HEK293T cells and purified with Strep-Tactin XT resin (IBA Lifesciences). Plasmids encoding BA.4/5 and BA.4+all RBD were transiently expressed in Expi293F™ Cells (ThermoFisher), cultured in FreeStyle™ 293 Expression Medium (ThermoFisher) at 30 °C with 8% CO₂ for 4 days. The harvested medium was concentrated using a QuixStand benchtop system. His-tagged RBDs were purified with a 5 mL HisTrap nickel column (GE Healthcare), followed by a Superdex 75 10/300 GL gel filtration column (GE Healthcare).

Liu C., Das R., Dijokaite-Guraliuc A., Zhou D., Mentzer A.J., Supasa P., Selvaraj M., Duyvesteyn H.M., Ritter T.G., Temperton N., Klenerman P., Dunachie S.J., Paterson N.G., Williams M.A., Hall D.R., Fry E.E., Mongkolsapaya J., Ren J., Stuart D.I, & Screaton G.R. (2024). Emerging variants develop total escape from potent monoclonal antibodies induced by BA.4/5 infection. Nature Communications, 15, 3284.

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