Nickel chelating chromatography

Manufactured by GE Healthcare

Sourced in United States

Nickel-chelating chromatography is a technique used for the purification and separation of proteins, particularly those containing a histidine tag. It utilizes the strong interaction between nickel ions and the histidine residues on the target protein to selectively capture and isolate it from a complex mixture. The core function of this technique is to provide a simple and efficient method for the purification of recombinant proteins, enabling researchers to obtain high-purity samples for further analysis and applications.

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Lab products found in correlation

Prset b vector, by Thermo Fisher Scientific (1 mentions) Amicon ultra 30 centrifugal filter, by Merck Group (1 mentions) Abspin trap protein g sepharose columns, by GE Healthcare (1 mentions) Lentil lectin affinity chromatography, by GE Healthcare (1 mentions) Freestyle 293 f suspension cells, by Thermo Fisher Scientific (1 mentions) Bira biotin ligase, by Avidity (1 mentions)

3 protocols using nickel chelating chromatography

Recombinant Vaccine Antigens Production

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The experimental vaccine consisted of an oil-in-water adjuvant combined with an alginate hydrogel (proprietary adjuvant, MSD-AH). As antigenic proteins, Efb and LukM, were used (50 μg/dose each). Cows were immunized with S. carnosus derived Efb and E. coli derived LukM. S. carnosus derived Efb was also used for ELISAs, while neutralization assays were performed with E. coli derived Efb, LukM and LukF’. For expression in S. carnosus, the gene encoding efb from the S. aureus Newbould305 strain (ATCC29740) was amplified by PCR and ligated into a pXR100 derived vector. S. carnosus culture supernatant was 0.2 μm filtered, analyzed on gel for Efb purity and concentration, and stored at −20 °C. For expression in E. coli, Efb, LukM and LukF’ proteins were generated as described previously [33 (link),34 (link)]. Briefly, the efb gene of the S. aureus Newman strain and the lukm and lukf gene sequences of the S. aureus field isolate S1444 were amplified by PCR and ligated into the pRSETB vector (Invitrogen). The proteins were expressed with a six-residue N-terminal HIS-tag and purified by nickel-chelating chromatography (GE Healthcare) according to the manufacturer’s manual. Purified proteins were dialyzed against PBS and stored at −20 °C.

Boerhout E., Vrieling M., Benedictus L., Daemen I., Ravesloot L., Rutten V., Nuijten P., van Strijp J., Koets A, & Eisenberg S. (2015). Immunization routes in cattle impact the levels and neutralizing capacity of antibodies induced against S. aureus immune evasion proteins. Veterinary Research, 46, 115.

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AE2F SOSIP.664 Nanoparticle Production

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A mammalian expression vector of AE2F SOSIP.664 was constructed by fusing a 6 × His-SpyTag sequence to the amino terminal end of AE2F SOSIP.664 sequence. The construct was transiently transfected into HEK293T cells, which were harvested 48 h later for purification of the soluble expressed 6 × His-SpyTag-AE2F SOSIP.664 proteins by nickel chelating chromatography (GE Health Care, Piscataway, NJ, USA) under native condition. VRC01 and 10E8 monoclonal antibodies were purified using Ab Spin Trap Protein G sepharose columns (GE Healthcare) following the manufacturer instructions. Protein purity was assessed by Coomassie-blue staining after separation denaturing gel electrophoresis followed by Coomassie blue staining, and the content of SpyTag-fused AE2F SOSIP.664 preparation was further analyzed by size exclusion chromatography. SpyCatcher-fused ferritin protein was kindly provided by Prof. Mingzhao Zhu (University of the Chinese Academy of Sciences, Beijing, China). The SOSIP.664-Ferritin nanoparticles were produced by mixing SOSIP.664-SpyTag with SpyCatcher-Ferritin at a molar ratio of 1:1, and then desalted on Amicon Ultra-30 centrifugal filter (Millipore, Billerica, MA, USA) into PBS.

Ding X., Cao K., Wang J., Wan Y., Chen Q., Ren Y., Zheng Y., Zhu M., Tian R., Wang W., Zhao C., Zhang X, & Xu J. (2021). Exploration of a Sequential Gp140-Gp145 Immunization Regimen with Heterologous Envs to Induce a Protective Cross-Reactive HIV Neutralizing Antibody Response In Non-human Primates. Virologica Sinica, 36(4), 784-795.

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Production and Purification of HIV-1 Env Proteins

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The soluble YU2-derived gp140-F trimers (36 (link)) used as the immunogen were produced by transient transfection into Freestyle 293F suspension cells (Invitrogen) as previously described (37 (link)). The Env ligands used in the binding studies were the following trimeric proteins: gp140-F, gp120-F, gp120-F-ΔV3, gp120-F-ΔV1V2, gp140-F-D368R, and the following monomeric proteins: TriMut, TriMut-368/370, TriMut-368/370/474 and gp140-GCN4 were purified by lentil-lectin and gel filtration chromatography. The biotinylated gp140-F probe used for single cell sorting by flow cytometry was purified by lentil-lectin affinity chromatography and nickel-chelating chromatography (GE Healthcare, Uppsala, Sweden). All probes carried an Avi-tag for site-specific biotinylation at the C-termini of the proteins and biotinylation was performed with biotin ligase Bir A (Avidity, Denver, CO). All Env proteins were from the YU2 strain except the TriMut proteins, which were from HXBc2. The collagen-foldon protein was kindly received from the laboratory of Professor Rikard Holmdahl at Karolinska Institutet, recombinant Ovalbumin (Ova) protein was purchased from Sigma-Aldrich and recombinant influenza hemagglutinin 1 (HA1) was produced as previously described (38 (link)).

Phad G.E., Bernat N.V., Feng Y., Ingale J., Murillo P.A., O’Dell S., Li Y., Mascola J.R., Sundling C., Wyatt R.T, & Karlsson Hedestam G.B. (2015). Diverse antibody genetic and recognition properties revealed following HIV-1 Env immunization. Journal of immunology (Baltimore, Md. : 1950), 194(12), 5903-5914.

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