Superose 6 increase

Manufactured by Cytiva

Superose 6 Increase is a size exclusion chromatography media designed for the purification of proteins, peptides, and other biomolecules. It features a high chemical and physical stability, allowing for repeated use. The media is available in different bead sizes to accommodate a variety of sample volumes and flow rates.

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Lab products found in correlation

Nanodrop 2000, by Thermo Fisher Scientific (2 mentions) Sinofection transfection reagent, by Sino Biological (1 mentions) Histrap excel 5 ml column, by Cytiva (1 mentions) Strep tactin superflow plus cartridge, by Qiagen (1 mentions) Superdex 200 pg, by Cytiva (1 mentions)

Close spelling variants of this product

Superose 6 Increase 10/300 Superose 6 Increase 3.2/300 Superose 6 Increase 3.2/300 column Superose 6 Increase 10/300 GL size-exclusion chromatography Superose 6 increase gel filtration column Superose 6 Increase 5/150 GL) Superose 6 Increase 10/300 column Superose 6 Increase 10/300 GL column Superose 6 Increase column Superose 6 Increase 5/150 GL column

5 protocols using superose 6 increase

Production and Purification of SARS-CoV-2 Spike Proteins

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All the constructs were transiently transfected into Expi293F^TM cells by Sinofection Transfection Reagent (Sino Biological, STF02) and cultured for 5 days.
The V regions of human mAbs were constructed on pCAGGS plasmid containing coding sequences for human IgG1 heavy chain or light chain. Antibodies were purified by AmMag^TM Protein A MagBeads (Genscript, L00672). Briefly, antibodies were combined with the beads, washed by AmMag^TM Wash buffer (Genscript, B00045) and eluted by 0.1 M Glycine. Medium was changed to PBS through centrifuge in concentration tubes (Merck millipore, UFC805008).
The coding sequences of SARS-CoV-2 prototype spike (GISAID: EPI_ISL_402119), Omicron BA.2 spike (GISAID: EPI_ISL_9652748), and Omicron BA.4/5 with a C-terminal 6× His tag and six mutations (F817P, A892P, A899P, A942P, K986P, V987P) were cloned into the pCAGGS vector, respectively. The cell supernatants expressing spike proteins were collected and purified by Ni affinity chromatography using a HisTrap^TM HP 5 mL column (Cytiva). The samples were further purified via gel filtration chromatography with Superose 6 Increase (Cytiva).

Guo S., Zheng Y., Gao Z., Duan M., Liu S., Du P., Xu X., Xu K., Zhao X., Chai Y., Wang P., Zhao Q., Gao G.F, & Dai L. (2023). Dosing interval regimen shapes potency and breadth of antibody repertoire after vaccination of SARS-CoV-2 RBD protein subunit vaccine. Cell Discovery, 9, 79.

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Purification of His-tagged Proteins

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Clarified and filtered culture media were applied to HisTrap Excel 5 ml column (Cytiva) at 3 ml/min followed by 10 CV wash with 50 mM sodium phosphate, 300 mM NaCl, 10 mM imidazole, pH 8.0. Bound proteins were eluted with 50 mM sodium phosphate, 300 mM NaCl, 500 mM imidazole, pH 8.0. Pooled eluate was loaded onto Strep-Tactin Superflow Plus cartridge (1 ml or 5 ml at 0.5 ml/min or 3 ml/min; Qiagen). The resin was washed with 10 CV of 50 mM sodium phosphate, 300 mM NaCl, pH 8.0 and proteins were eluted in the same buffer with 2.5 mM desthiobiotin. Fractions containing protein were pooled, concentrated in 30 kDa MWCO concentrator (Pierce) to 0.1-0.4 ml and manually injected onto Superose 6 Increase (Cytiva) equilibrated in 20 mM Hepes, 150 mM NaCl, pH 7.6. Protein concentration was determined using Nanodrop 2000 or 8000 (Thermo Fisher).

Riley T.P., Chou H.T., Hu R., Bzymek K.P., Correia A.R., Partin A.C., Li D., Gong D., Wang Z., Yu X., Manzanillo P, & Garces F. (2021). Enhancing the Prefusion Conformational Stability of SARS-CoV-2 Spike Protein Through Structure-Guided Design. Frontiers in Immunology, 12, 660198.

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Radiolabeling and Serum Stability of αCD11b Antibody

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⁸⁹Zr-DFO-αCD11b was prepared and its purity determined by SEC-HPLC as previously described [24] . For radiolabelling of Lumi804-αCD11b, the antibody (10 nmol, 187 µL) was added to Zr-89 oxalate (111 MBq, 20 µL) or Lu-177 chloride (111 MBq, 7 µL) in 0.5 M ammonium acetate buffer pH 5.0 (200 µL) with gentisic acid (56 mM stock solution, 2 µL) and incubated for 30 min at 25 °C (Fig. 1). For all radiolabelled antibody conjugates, determination of radiochemical yield was performed by first diluting a small aliquot of the reaction mixture with 50 mM DTPA, pH 5.5, and then monitoring the reaction progress by SEC-HPLC (Superose, PBS, 0.6 mL/min). Radiochemical yield for all conjugates was ≥ 99% with a molar activity of 11.1 GBq/µmol. Radiolabelling with high molar activities 22.2 GBq/µmol and 33.3 GBq/µmol was also achieved for ¹⁷⁷Lu-Lumi804-αCD11b with ≥ 99% radiochemical yield. Serum stability of both ⁸⁹Zr- or ¹⁷⁷Lu-Lumi804-αCD11b Lumi804-αCD11b (n=2 each) was monitored by radio-SEC-HPLC (Superose 6 Increase (Cytiva), 10/300, 1 mL/min, PBS) over time (24, 48, 72, 96 h) in mouse serum. For in vivo studies, the radiolabelled antibody conjugates were diluted in the sterile saline before i.v. injections.

Foster A., Nigam S., Tatum D.S., Raphael I., Xu J., Kumar R., Plakseychuk E., Latoche J.D., Vincze S., Li B., Giri R., McCarl L.H., Edinger R., Ak M., Peddagangireddy V., Foley L.M., Hitchens T.K., Colen R.R., Pollack I.F., Panigrahy A., Magda D., Anderson C.J., Edwards W.B, & Kohanbash G. (2021). Novel theranostic agent for PET imaging and targeted radiopharmaceutical therapy of tumour-infiltrating immune cells in glioma. EBioMedicine, 71, 103571.

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Expression and Purification of Brpt5.5

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Brpt5.5 constructs were generated from the wild-type plasmid previously used, containing an N-terminal, TEVp-cleavable His-MBP tag [16 (link), 20 (link)], and the H85A mutants were generated from primers listed previously [20 (link)]. A C-terminal, TEVp-cleavable Strep-II Tag for SAXS experiments was added with a glycine linker (-G-) following the C-terminal half-repeat of Brpt5.5 (…EYGPT-G-ENLYFQ-GWSHPQFEK). Non-Strep-tagged proteins were expressed and purified as previously described [16 (link)]. For purification of the Strep-tagged proteins, a Strep-affinity purification step was added after the initial Ni-affinity using a Cytiva StrepTrap HP (5mL) column. After TEVp-cleavage, a subtractive Ni-affinity step was used to remove uncleaved fusion protein and His-MBP. Next, a subtractive StrepTrap step removed uncleaved Strep-tagged fusion protein and Strep tag. Lastly, a Superdex 200 pg (Cytiva) or Superose 6 Increase (Cytiva) was used as a final purification step. For all experiments (except where denoted explicitly), protein was dialyzed into 50 mM MOPS pH 7.2, 50 mM NaCl.

Yarawsky A.E., Hopkins J.B., Chatzimagas L., Hub J.S, & Herr A.B. (2022). Solution structural studies of pre-amyloid oligomer states of the biofilm protein Aap. Journal of molecular biology, 434(16), 167708.

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Reconstitution of Nucleosomes with Modified Histones

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Nucleosome containing wild-type or modified histones were reconstituted as previously reported (46 ). Briefly, histone H2A/H2B dimers and H3/H4 tetramers were prepared from individual histone proteins, by dissolving the combined histones in an unfolding buffer [6 M Gn-HCl and 25 mM Hepes (pH 7.5)] and dialyzing the mixture with refolding buffer [2 M NaCl and 25 mM Hepes (pH 7.5)]. The homogeneous dimer and tetramers were isolated by size exclusion chromatography. Nucleosomes were prepared by mixing histone dimers, tetramers, and DNA601 (dimer:tetramer:DNA = 2:1:1) under high salt buffer [2 M KCl and 10 mM Hepes (pH 7.5)] followed by salt gradient dialysis to 10 mM KCl over 20 hours. The nucleosomes were purified by size exclusion chromatography (Superose 6 increase 3.2/300 or 10/300, Cytiva).

Liu Z., Wu Y., Mao X., Kwan K.C., Cheng X., Li X., Jing Y, & Li X.D. (2023). Development of multifunctional synthetic nucleosomes to interrogate chromatin-mediated protein interactions. Science Advances, 9(18), eade5186.

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