Hiload superdex200 column

Manufactured by Cytiva

The HiLoad Superdex200 column is a size exclusion chromatography column designed for the purification and analysis of biomolecules. It is capable of separating a wide range of proteins, peptides, and other macromolecules based on their molecular size and shape.

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Lab products found in correlation

Source s cation exchange chromatography, by Cytiva (2 mentions) Talon resin, by Takara Bio (2 mentions) Hitrap q column, by Cytiva (2 mentions) Completetm his tag purification resin, by Roche (1 mentions) Dithiothreitol (dtt), by Merck Group (1 mentions)

3 protocols using hiload superdex200 column

Purification and Oxidation/Reduction of PDI Variants

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The expression and purification of the PDI-b’a’ (residues 208–449) and PDI-b’a’ ^C365S/C368S from Humicola insolens were performed as previously described [8 (link),11 (link)]. YFP-PDI-b’a’-CFP and their variants were expressed with an N-terminal hexahistidine tag using the pCold-I vector in E. coli and purified using a Ni²⁺-immobilized affinity column (cOmplete^TM His-Tag Purification Resin, Roche) from the soluble lysate. After the cleavage of the hexahistidine tag with TEV protease, these proteins were purified using a HiLoad Superdex 200 column (Cytiva) in 50 mM Tris–HCl (pH 8.0).
To oxidize the a’ active site, the purified protein (1 mg/mL) was dialyzed using 50 mM Tris-HCl (pH 8.0) containing 0.1 mM oxidized glutathione for 10 days. To reduce the a’ active site, the protein was dissolved in a buffer containing 2–4 mM DTT (Sigma Aldrich, St. Louis, MO, USA).

Yagi-Utsumi M., Miura H., Ganser C., Watanabe H., Hiranyakorn M., Satoh T., Uchihashi T., Kato K., Okazaki K.I, & Aoki K. (2023). Molecular Design of FRET Probes Based on Domain Rearrangement of Protein Disulfide Isomerase for Monitoring Intracellular Redox Status. International Journal of Molecular Sciences, 24(16), 12865.

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Production and Purification of SIRT6 and SNF2h

Cited 1 time

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The gene coding for the full-length human SIRT6 (UniProtKB: Q8N6T7) was cloned into pST50Tr (34 (link)) vector with an N-terminal Gly-Ser-Ser-hexahistidine (His₆). Gly-Ser-Ser-(His₆)-SIRT6 was expressed in BL21(DE3) pLysS E. coli cells at 23°C. Bacterial cells were lysed by sonication, and the crude lysate was centrifuged at 36,000g for 40 min at 4°C. The protein was purified by metal affinity chromatography (TALON resin, Clontech), the affinity tag was removed using tobacco etch virus (TEV) protease, and the protein was further purified by Source S cation-exchange chromatography (Cytiva).
His₆-tagged human SNF2h was expressed and purified as previously described with minor modifications (35 (link)). Briefly, His₆-SNF2h was expressed in BL21(DE3) Rosetta E. coli cells at 18°C. Cells were lysed via sonication, and Ni–nitrilotriacetic acid affinity chromatography was used to isolate His₆-SNF2h from the clarified lysate. TEV protease was used to remove the His₆-tag, and the untagged SNF2h was passed through a HiTrapQ column (Cytiva) to remove contaminating DNA. The protein was then run over a HiLoad Superdex200 column (Cytiva), and pure fractions were pooled, aliquoted, and stored at −80°C.

Chio U.S., Rechiche O., Bryll A.R., Zhu J., Leith E.M., Feldman J.L., Peterson C.L., Tan S, & Armache J.P. (2023). Cryo-EM structure of the human Sirtuin 6–nucleosome complex. Science Advances, 9(15), eadf7586.

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Expression and Purification of SIRT6 and SNF2h

Cited 1 time

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The gene coding for the full-length human SIRT6 (UniProtKB: Q8N6T7) was cloned into pST50Tr (35 (link)) vector with an N-terminal GSS-hexahistidine (His₆). GSS-(His₆)-SIRT6 was expressed in BL21(DE3) pLysS Escherichia coli cells at 23°C. Bacterial cells were lysed by sonication, and the crude lysate centrifuged at 36,000 g for 40 min at 4°C. The protein was purified by metal-affinity chromatography (Talon resin, Clontech), the affinity tag removed using tobacco etch virus (TEV) protease and the protein further purified by Source S cation-exchange chromatography (Cytiva).
His₆-tagged human SNF2h was expressed and purified as previously described with minor modifications (36 (link)). Briefly, His₆-SNF2h was expressed in BL21(DE3) Rosetta Escherichia coli cells at 18°C. Cells were lysed via sonication, and Ni-NTA affinity chromatography used to isolate His₆-SNF2h from the clarified lysate. TEV protease was used to remove the His₆-tag, and the untagged SNF2h was passed through a HiTrapQ column (Cytiva) to remove contaminating DNA. The protein was then run over a HiLoad Superdex200 column (Cytiva), and pure fractions were pooled, aliquoted, and stored at −80 °C.

Chio U.S., Rechiche O., Bryll A.R., Zhu J., Feldman J.L., Peterson C.L., Tan S, & Armache J.P. (2023). Cryo-EM structure of the human Sirtuin 6-nucleosome complex. bioRxiv.

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