Superdex 200 10 300 gl gel filtration column

Manufactured by GE Healthcare

Sourced in United States, Sweden, United Kingdom

The Superdex 200 10/300 GL gel filtration column is designed for the separation and purification of proteins, peptides, and other biomolecules. It is a size-exclusion chromatography column that separates molecules based on their size and shape. The column is packed with a highly cross-linked agarose-dextran matrix, which provides a wide separation range and high resolution. The Superdex 200 10/300 GL column has a bed volume of 24 ml and can be used for a wide variety of applications in the field of biochemistry and biotechnology.

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Lab products found in correlation

Pe streptavidin, by Merck Group (3 mentions) Astra 6, by Wyatt Technology (2 mentions) Vitrobot mark 4, by Thermo Fisher Scientific (2 mentions) High performance liquid chromatography pump, by Agilent Technologies (1 mentions) D biotin, by Avidity (1 mentions) Akta purifier fplc system, by GE Healthcare (1 mentions) Minidawn treos three angle light scattering detector, by Wyatt Technology (1 mentions) Optilab t rex refractive index detector, by Wyatt Technology (1 mentions) Biacore t200, by GE Healthcare (1 mentions) Biacore 3000, by GE Healthcare (1 mentions) Sensor cm5, by GE Healthcare (1 mentions) Kta fplc system, by Cytiva (1 mentions) Protease inhibitor mixture, by Roche (1 mentions) Facscalibur flow cytometer, by BD (1 mentions) Sf9 insect cells, by Thermo Fisher Scientific (1 mentions) Hi5 insect cells, by Thermo Fisher Scientific (1 mentions) Resource q anion exchange column, by GE Healthcare (1 mentions) Ultracel 10k centrifugal filter, by Merck Group (1 mentions) Ni nta bead, by Qiagen (1 mentions) Simplyblue safestain, by Thermo Fisher Scientific (1 mentions)

24 protocols using superdex 200 10 300 gl gel filtration column

SEC-MALS Analysis of MexT Proteins

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The sizes of FL MexT and MexT RD were assayed using SEC-MALS. A high-performance liquid chromatography pump (Agilent, USA) was connected to a Superdex-200 10/300 GL gel filtration column (GE Healthcare) and a MALS instrument (Wyatt Dawn Heleos, USA). The size-exclusion chromatography column was pre-equilibrated with buffer comprising 20 mM Tris-HCl (pH 8.5), 500 mM NaCl, and 2 mM 2-mercaptoethanol for FL MexT; and 20 mM Tris-HCl (pH 8.5), 300 mM NaCl, and 2 mM 2-mercaptoethanol for MexT RD. Bovine serum albumin (2 mg/ml) was used as the standard. MexT RD and FL MexT samples (2 mg/ml) were injected onto the column and eluted at a flow rate of 0.2 ml/min. The datasets were evaluated using the Debye model for fitting static light-scattering data, and refractive index peaks were presented in EASI graphs created using Astra V software (Wyatt Dawn Heleos).

Kim S., Kim S.H., Ahn J., Jo I., Lee Z.W., Choi S.H, & Ha N.C. (2019). Crystal Structure of the Regulatory Domain of MexT, a Transcriptional Activator of the MexEF-OprN Efflux Pump in Pseudomonas aeruginosa. Molecules and Cells, 42(12), 850-857.

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HLA-A*0201 Tetramer Preparation for Peptide Presentation

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Human leukocyte antigen-A^*0201-restricted tetramers with peptide NY-ESO-1_157−165: SLLMWITQC or HPV-E6₂₉₋₃₈: TIHDIILECV were prepared as previously described (48 (link)). Briefly, the extracellular domain of HLA-A^*0201 (1-276) was modified by the addition of a substrate sequence for the biotinylation enzyme BirA at the C-terminus of the α3 domain. The modified HLA-A2 and β2-microglobulin were expressed in Escherichia coli and refolded in the presence of peptides. In vitro-renatured peptide/HLA-A2 complexes were then purified and biotinylated by incubation with D-biotin, ATP, and the biotin protein ligase BirA (Avidity, Aurora, Colorado) at 4°C for 12 h. The samples were further purified using a Superdex 200 10/300 GL gel filtration column (GE Healthcare, Chicago, Illinois). Finally, the tetramers were formed by mixing with PE-streptavidin (Sigma-Aldrich, Saint Louis, Missouri) and stored at 4°C in PBS containing 0.5 mM EDTA, 0.2% BSA, 10 mM Tris-HCI (pH 8.0), 150 mM NaCI, and 0.09% NaN₃.

Zhang H., Sun M., Wang J., Zeng B., Cao X., Han Y., Tan S, & Gao G.F. (2021). Identification of NY-ESO-1157–165 Specific Murine T Cell Receptors With Distinct Recognition Pattern for Tumor Immunotherapy. Frontiers in Immunology, 12, 644520.

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Structural Determination of PrgB Dimer

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PrgB_188–1233 dimer fractions were thawed on ice and loaded on a Superdex 200 10/300 GL gel filtration column (GE Healthcare) equilibrated in 20 mM HEPES/NaOH pH 7.0 and 150 mM NaCl. Protein peak fractions, corresponding to the dimer, were diluted to 0.1–0.3 mg/ml and for the DNA-bound structures 120-bp ssDNA (Table 1) was added in 1:1.2 molar ratio (protein:DNA) and samples were incubated for 15 min on ice. For both apo and DNA-bound samples, 4 μl of sample was applied to glow discharged Quantifoil 300 mesh 1.2/1.3 (Quantifoil) grids at 4°C and 90–100% humidity, blotted for 1 s with blot force −5, and plunge-frozen in liquid ethane using a Vitrobot Mark IV (Thermo Fisher Scientific).

Sun W.S., Lassinantti L., Järvå M., Schmitt A., ter Beek J, & Berntsson R.P. (2023). Structural foundation for the role of enterococcal PrgB in conjugation, biofilm formation, and virulence. eLife, 12, RP84427.

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Gel Filtration Purification of TRX-T-TXNIP

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A Superdex 200 10/300 GL gel filtration column (GE Healthcare) installed on an AKTA purifier FPLC system (GE Healthcare) was equilibrated with 50 mM Tris-HCl (pH 8.0), 500 mM NaCl and 10% glycerol at a flow rate of 0.4 ml min⁻¹ at room temperature. The purified TRX–T–TXNIP complex and its mutants were injected onto the column. Ovalbumin was used as the molecular weight standard.

Hwang J., Suh H.W., Jeon Y.H., Hwang E., Nguyen L.T., Yeom J., Lee S.G., Lee C., Kim K.J., Kang B.S., Jeong J.O., Oh T.K., Choi I., Lee J.O, & Kim M.H. (2014). The structural basis for the negative regulation of thioredoxin by thioredoxin-interacting protein. Nature Communications, 5, 2958.

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Purification and Characterization of IMP3 RRM12

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Purified IMP3 RRM12 (in buffer 20 mM Tris pH 7.5, 150 mM NaCl, 1 mM DTT) was concentrated to 5 mg ml⁻¹ and loaded to a Superdex 200 10/300 GL gel-filtration column (GE Healthcare) equilibrated in 20 mM Tris pH 7.5, 200 mM NaCl, 0.02% w/v NaN₃, and 1 mM DTT. Light scattering was recorded with an in-line miniDAWN TREOS three angle light scattering detector (Wyatt Technology), and protein concentration was detected using an in-line Optilab T-rEX refractive index detector (Wyatt Technology). The molecular mass of IMP3 RRM12 was calculated using ASTRA 6 software (Wyatt Technology).

Jia M., Gut H, & Chao J.A. (2018). Structural basis of IMP3 RRM12 recognition of RNA. RNA, 24(12), 1659-1666.

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Characterizing sDM-Fab Interactions

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Experiments with sDM immobilized were performed at 25 °C, using a Biacore 3000 (GE Healthcare). Experiments with Fab immobilized were performed using a Biacore T200 (GE Healthcare). Stock solutions of sDM and b12 Fab were monodisperse, after purification on a Superdex 200 10/300 GL gel filtration column (GE Healthcare). sDM-biotin was immobilized to ~1000 RUs onto a streptavidin-coated (SA) sensor (GE Healthcare) by injection of ~10 nM at 10 μl/min in HEPES-buffered saline with surfactant P20 (HBS-P). b12 Fab was immobilized to ~4000 RUs, by amine chemistry, onto a CM5 sensor (GE Healthcare), injecting at 5 μl/min, 2.5 μg/ml, in pH 5, 10 mM sodium acetate buffer. sDM/Fab interaction was measured in 20mM MES (pH 5.7 or pH 6.7), 100mM NaCl, 0.002% p20). Where indicated, NaCl was elevated to 150 mM. Varying concentrations of sDM, b12 Fab or 40 μM Fc were injected (20 μl/min) across a reference flow cell and the flow cell with immobilized ligand. To determine the apparent K_D, the reference signal was subtracted at each time point, and equilibrium data were fit to the “one site-specific” mass action model using Prism 6 software.

Macmillan H., Strohman M.J., Ayyangar S., Jiang W., Rajasekaran N., Spura A., Hessell A.J., Madec A.M, & Mellins E.D. (2014). The MHC class II cofactor, HLA-DM, interacts with immunoglobulin in B cells. Journal of immunology (Baltimore, Md. : 1950), 193(6), 2641-2650.

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Sizing of (+)-Ligand Substrate by SEC

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Size-exclusion chromatography (SEC) was conducted using an Äkta FPLC system (Amersham Biosciences, Uppsala, Sweden) equipped with a Superdex-200 10/300 GL gel filtration column (GE Healthcare Life Sciences); 300 μL of 100 μM (+)-LS was loaded onto the column that was equilibrated in 50 mM Tris (pH 8), 100 mM NaCl, and 10% glycerol at 4 °C. The flow was maintained at a constant rate of 0.5 mL/min. The void volume was measured at 8.3 mL by eluting blue dextran as a high-molecular weight standard (MW ~ 2000 kDa). The total internal column volume was measured at 21.19 mL with vitamin B₁₂ as a low-molecular weight standard (MW = 1.35 kDa). These and other well-resolved molecular weight standards were used to derive a standard curve that allowed accurate estimation of the molecular weight of eluted (+)-LS.

Morehouse B.R., Kumar R.P., Matos J.O., Olsen S.N., Entova S, & Oprian D.D. (2017). Functional and Structural Characterization of a (+)-Limonene Synthase from Citrus sinensis. Biochemistry, 56(12), 1706-1715.

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Protein Fractionation and Analysis

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After treatments, PC3 cells were collected and homogenized in homogenization buffer (40 mM Tris-HCl pH 7.5, 150 mM NaCl and a protease inhibitor mixture from Roche Applied Science) by repeatedly passing (15 times) through a 1 ml syringe with a 23-gauge needle. The homogenate was centrifuged at 13,000g for 30 min. The resulting supernatants (2.0 mg protein in 500 μl) were then applied to a Superdex 200 10/300 GL gel filtration column (GE Healthcare) and eluted at a flow rate of 0.5 ml min⁻¹ with 40 mM Tris-HCl, pH 7.5 and 150 mM NaCl. Fractions of 500 μl were collected and 20 μl from each fraction was analysed by western blot.

Zhang F., Kumano M., Beraldi E., Fazli L., Du C., Moore S., Sorensen P., Zoubeidi A, & Gleave M.E. (2014). Clusterin facilitates stress-induced lipidation of LC3 and autophagosome biogenesis to enhance cancer cell survival. Nature Communications, 5, 5775.

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Peptide-MHC Tetramer Preparation and Staining

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H-2D^b-restricted tetramers of peptides NP_366−374 and PA_224−233 and HLA-A*24-restricted tetramers of peptides H1-P25 and H7-P25 were prepared as previously described (30 (link)). Preparation and staining of H-2D^b-restricted tetramers were performed as follows. Briefly, to produce biotinylated peptide-MHC protein, H-2D^b was modified by the addition of a substrate sequence for biotinylating enzyme BirA at the C terminus of the α3 domain. In vitro-renatured H-2D^b/peptide complexes were then purified and biotinylated by incubation with d-biotin, ATP, and the biotin protein ligase BirA (Avidity) at 4°C for 12 h. The biotinylated H-2Db was further purified using a Superdex 200 10/300 GL gel-filtration column (GE Healthcare) to remove excess biotin and then mixed with PE-streptavidin (Sigma). Cells from the subjects were stained with PE-tetramer, FITC-conjugated anti-CD3 antibody, and PerCP-cy5.5 anti-CD8 antibody. All samples were analyzed with a FACSCalibur flow cytometer (Becton, Dickinson) after staining.

Zhao M., Liu K., Luo J., Tan S., Quan C., Zhang S., Chai Y., Qi J., Li Y., Bi Y., Xiao H., Wong G., Zhou J., Jiang T., Liu W., Yu H., Yan J., Liu Y., Shu Y., Wu G., Wu A., Gao G.F, & Liu W.J. (2018). Heterosubtypic Protections against Human-Infecting Avian Influenza Viruses Correlate to Biased Cross-T-Cell Responses. mBio, 9(4), e01408-18.

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Expressing and Purifying IEkMCC Protein

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Plasmid construct for expressing the extracellular domain of IEk with covalently linked MCC peptide was a gift from John Kappler (National Jewish Health). For conjugation with polymers, the construct was modified by PCR to add a cysteine at the C-terminus of the β chain. Baculoviruses were generated with the construct and BaculoGold Linearized baculovirus DNA (BD Biosciences, San Diego, CA) in Sf9 insect cells (Invitrogen, Calsbad, CA). High titer stocks of cloned recombinant baculovirus were used to infect Hi5 insect cells (Invitrogen, Calsbad, CA) cultured in spinner flasks. IEkMCC protein was purified from the supernatant of infected Hi5 cell cultures using an affinity chromatography column conjugated with 14-4-4s antibody. The protein was further purified using a Superdex 200 10/300 GL gel filtration column (GE Healthcare, Piscataway, NJ) before conjugating with PEG linkers. The design and production of IEkMCC with a C-terminal AviTag was described previously [15] (link). The protein was biotinylated at the lysine residue of the AviTag using BirA enzyme as described previously [15] (link).

Ma Z., LeBard D.N., Loverde S.M., Sharp K.A., Klein M.L., Discher D.E, & Finkel T.H. (2014). TCR Triggering by pMHC Ligands Tethered on Surfaces via Poly(Ethylene Glycol) Depends on Polymer Length. PLoS ONE, 9(11), e112292.

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