Superdex 200

Manufactured by Bio-Rad

Sourced in United States

Superdex 200 is a size exclusion chromatography medium designed for high-resolution separation of proteins, peptides, and other biomolecules. It is composed of cross-linked agarose and dextran beads, providing a porous structure for size-based separation. The medium has a fractionation range suitable for molecules with molecular weights between 10,000 and 600,000 Daltons.

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Lab products found in correlation

Superdex 200, by GE Healthcare (2 mentions) Gel filtration standard, by Bio-Rad (1 mentions) 10dg column, by Bio-Rad (1 mentions) Biologic system, by Bio-Rad (1 mentions) A gel filtration standard, by Bio-Rad (1 mentions) Biologic duoflow, by Bio-Rad (1 mentions)

5 protocols using superdex 200

Oligomeric State Characterization of Protein

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The oligomeric state of the protein in solution was determined as a function of concentration and temperature. Size exclusion chromatography (SEC) was applied to confirm the homotetrameric state in the concentration range from 1 to 10 mg mL⁻¹ at a constant, room temperature. The experiments were carried out using a Superdex 200 gel filtration column pre-equilibrated with buffer B and calibrated with a gel filtration standard (BioRad).
Dynamic light scattering (DLS) experiments were performed to establish the oligomeric state of the enzyme at various temperatures but at a constant concentration of 5 mg mL⁻¹. Three scenarios were utilized: (i) unheated protein sample measured at 298 K; (ii) protein sample heated to 358 K for 30 min and then measured at 298 K; and (iii) protein sample heated to 358 K for 30 min and then measured at 358 K. All samples were filtered (0.1 μm) prior to the experiment.

Brzezinski K., Czyrko J., Sliwiak J., Nalewajko-Sieliwoniuk E., Jaskolski M., Nocek B, & Dauter Z. (2017). S-adenosyl-L-homocysteine hydrolase from a hyperthermophile (Thermotoga maritima) is expressed in Escherichia coli in inactive form – Biochemical and structural studies. International journal of biological macromolecules, 104(Pt A), 584-596.

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Reconstitution of Iron-Sulfur Clusters

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Reconstitution was performed on ice in an anaerobic chamber. Either full-length Asp1 or Asp1^371–920 was combined with 4 equiv of both ferric citrate and (NH₄)₂Fe(SO₄)₂ and 8 equiv of Na₂S under anaerobic conditions in buffer containing 40 mM HEPES (pH 7.2), 100 mM NaCl and 50 mM DTT. After 30 min, samples were removed from the anaerobic chamber and applied to a 10DG column (Bio-Rad) and Superdex200 to separate the protein from excess iron and sulfide. Protein was then concentrated and stored at −80 °C. Controls were performed which did not contain either Fe or S, as indicated in the figure legends.

Wang H., Nair V.S., Holland A.A., Capolicchio S., Jessen H.J., Johnson M.K, & Shears S.B. (2015). Asp1 from Schizosaccharomyces pombe Binds a [2Fe-2S]2+ Cluster Which Inhibits Inositol Pyrophosphate 1-Phosphatase Activity. Biochemistry, 54(42), 6462-6474.

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Recombinant Nsp7 Protein Purification

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The recombinant Nsp7 protein was purified by immobilized-metal affinity chromatography (IMAC) using a polyhistidine tag and further purified by a gel filtration column Superdex200 (GE Healthcare, Sweden). The cell pellet was suspended and lysed by sonication on ice. The lysate was centrifuged at 16,000 × g for 30 min, and the supernatant was collected and transferred to a Ni-NTA His Band Resin column pre-equilibrated with binding buffer (500 mM NaCl, 20 mM Tris, 5 mM imidazole). More than five column-volumes of washing buffer (500 mM NaCl, 20 mM Tris, 20 mM imidazole) was added to remove the nonspecific binding proteins. The target protein was eluted with elution buffer (500 mM NaCl, 20 mM Tris, 400 mM imidazole). The purity and relative concentration of the recombinant Nsp7 was determined by SDS-PAGE. The protein was further fractionated by gel filtration on a column of Superdex200 in a buffer of 50 mM Tris, 150 mM NaCl by using the Bio-Rad BioLogic system (Bio-Rad Laboratories, USA). The protein of interest was collected in different fractions according to its different states of aggregation. The final protein products were examined by SDS-PAGE before storing at −80℃.

Li H., Yang J., Bao D., Hou J., Zhi Y., Yang Y., Ji P., Zhou E., Qiao S, & Zhang G. (2017). Development of an immunochromatographic strip for detection of antibodies against porcine reproductive and respiratory syndrome virus. Journal of Veterinary Science, 18(3), 307-316.

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Oligomerization States of GST-A3H Variants

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The oligomerization states of GST-A3H (WT, Y112A/Y113A, W115A, and R175E/R176E) were determined by SEC using 10 μg of purified protein. The column (0.5 cm diameter and 16 cm height) was prepared by pouring a 10 mL Superdex 200 (GE Healthcare) resin bed. The running buffer contained 20 mM Tris, pH 8.0, 300 mM NaCl, 10% glycerol, and 5 mM DTT. A gel filtration standard (Bio-Rad) was used to generate a calibration curve from which the apparent molecular weights of eluted proteins were determined. The column was run using a Bio-Rad BioLogic DuoFlow, however, due to the low amount of protein applied, the chromatograms from the 10 ml Superdex 200 column were constructed by analyzing the integrated gel band intensities of the protein in each fraction after resolution by SDS-PAGE. The gels for each panel were resolved, stained with Oriole fluorescent gel stain, and scanned in parallel.

Feng Y., Wong L., Morse M., Rouzina I., Williams M.C, & Chelico L. (2018). RNA-mediated dimerization of the human deoxycytidine deaminase APOBEC3H influences enzyme activity and interaction with nucleic acids. Journal of molecular biology, 430(24), 4891-4907.

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SAXS Analysis of Molecular Models

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SAXS measurements were performed at the Advanced Photon Source at Argonne National Laboratory (beamline 18-ID) with an in-line SEC columns (Superdex 200 or Biorad EnRich 5–650 10–300) equilibrated with 20 mM HEPES, pH 7.4, and 150 mM NaCl. Data were analyzed using autorg and datgnom using the commands “autorg –sminrg 0.55 –smaxrg 1.1” and “datgnom ‘1’.dat -r ‘2’ – skip ‘3’ -o ‘1’.out,” respectively, where ‘1’ is the file name, ‘2’ is the R_g determined by autorg, and ‘3’ is the number of points removed at low q as determined from autorg. SAXS curves of molecular models were generated with Crysol version 2.83^{88 (link)}.

Leon K., Cunningham R.L., Riback J.A., Feldman E., Li J., Sosnick T.R., Zhao M., Monk K.R, & Araç D. (2020). Structural basis for adhesion G protein-coupled receptor Gpr126 function. Nature Communications, 11, 194.

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