Ni nta imac sepharose 6 fast flow resin

Manufactured by GE Healthcare

Ni-NTA IMAC Sepharose 6 Fast Flow resin is a pre-packed affinity chromatography medium designed for the purification of recombinant proteins containing a polyhistidine (His) tag. The resin consists of agarose beads that are coupled with nickel-nitrilotriacetic acid (Ni-NTA) ligands, which selectively bind to the His-tagged proteins. The fast flow properties of the resin allow for efficient and high-throughput purification of the target proteins.

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Lab products found in correlation

Superdex 200 column, by GE Healthcare (5 mentions) Polyethyleneimine, by Polysciences (5 mentions) Hek293 freestyle cells, by Thermo Fisher Scientific (4 mentions) Serum free media, by Thermo Fisher Scientific (3 mentions) Superdex 200, by GE Healthcare (1 mentions) Superdex 75 26 600, by GE Healthcare (1 mentions) Freestyle 293 f, by Thermo Fisher Scientific (1 mentions) Superdex 200 26 600, by GE Healthcare (1 mentions) Endoproteinase lys c, by Merck Group (1 mentions) Polyethylenimine (pei), by Polysciences (1 mentions)

7 protocols using ni nta imac sepharose 6 fast flow resin

SARS-CoV-2 Spike Protein and Fab Production

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Recombinant SARS-CoV-2 S2P spike was produced as previously described^{23 (link),24 (link)}. Briefly, protein was expressed in HEK 293 Freestyle cells (Invitrogen, Carlsbad, CA) in suspension culture using serum-free media (Invitrogen) by transient transfection using polyethyleneimine (Polysciences, Warrington, PA). Four days after transfection, the secreted protein was purified by nickel affinity chromatography using Ni-NTA IMAC Sepharose 6 Fast Flow resin (GE Healthcare, Chicago, IL) followed by size exclusion chromatography on a Superdex 200 column (GE Healthcare) in 10 mM Tris, 150 mM NaCl, pH 7.4.
Monoclonal antibody LP5 was digested using a previously published protocol^{33 (link)} to generate the Fab fragment. Briefly, IgG was digested with immobilized papain at 37 °C for 3 hrs in 50 mM phosphate buffer, 120 mM NaCl, 30 mM cysteine, 1 mM EDTA, pH 7. The resulting Fab was purified from Fc by affinity chromatography using protein A resin. Fab purity was analyzed by SDS-PAGE and buffer-exchanged into 10 mM Tris, 150 mM, pH 7.4 for cryo-EM experiments.

Madan B., Reddem E.R., Wang P., Casner R.G., Nair M.S., Huang Y., Fahad A.S., de Souza M.O., Banach B.B., López Acevedo S.N., Pan X., Nimrania R., Teng I., Bahna F., Zhou T., Zhang B., Yin M.T., Ho D.D., Kwong P.D., Shapiro L, & DeKosky B.J. (2021). Antibody screening at reduced pH enables preferential selection of potently neutralizing antibodies targeting SARS‐CoV‐2. Aiche Journal. American Institute of Chemical Engineers, 67(12), e17440.

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Purification of SARS-CoV-2 Receptor-Binding Domain

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The SARS-CoV-2 RBD (residues 331-541), was cloned into the pVRC-8400 mammalian expression plasmid, with a C-terminal 6X-His-tag and an intervening HRV-3C protease cleavage site. Expression vector was transiently transfected into Expi293F GnTI^-cells suspension culture in serum-free media (Invitrogen). Media was harvested 6 days after transfection and the secreted protein purified using Ni-NTA IMAC Sepharose 6 Fast Flow resin (GE Healthcare) followed by size exclusion chromatography (SEC) on Superdex 200 (GE Healthcare) in 10 mM, Tris pH 8.0, 150 mM NaCl (SEC buffer). For enzymatic deglycosylation of RBD was carried out by adding 1.0 mL Endo Hf (NEB) per 10 mg of RBD and incubating for 24 h at 4°C; a second round of SEC was performed to remove excess Endo Hf. Protein purity was analysed by SDS-PAGE at every step.

Luo M., Zhou B., Reddem E.R., Tang B., Chen B., Zhou R., Liu H., Liu L., Katsamba P.S., Au K.K., Man H.O., To K.K., Yuen K.Y., Shapiro L., Dang S., Ho D.D, & Chen Z. (2023). Structural insights into broadly neutralizing antibodies elicited by hybrid immunity against SARS-CoV-2. Emerging Microbes & Infections, 12(1), 2146538.

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Recombinant Antibody Fab Production

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Recombinant antibody Fabs were transiently expressed in FreeStyleTM 293F (Invitrogen) suspension cultures by co-transfection of pVRC8400 plasmids containing expression constructs for light chain and Fab heavy chain using polyethyleneimine (Polysciences). Cell growth was harvested on day 6 post transfection. eOD-GT6 was also produced in FreeStyleTM 293F (Invitrogen) suspension cultures by transient transfection using polyethyleneimine (Polysciences) of a pHLSec plasmid containing mammalian codon-optimized eOD-GT6 with a C-terminal Avi and His6x affinity tag. Proteins were harvested from the supernatant after 96 h.
The secreted proteins were purified by using Ni-NTA IMAC Sepharose 6 Fast Flow resin (GE Healthcare) nickel affinity chromatography followed by size exclusion chromatography (SEC) using a Superdex 200 26/600 (Fabs) or Superdex 75 26/600 (eOD) column (GE Healthcare) in 10 mM Tris pH 8.0, 150 mM NaCl SEC buffer. Peak fractions containing Fabs or eOD-GT6 were pooled. Protein purity was analyzed by SDS-PAGE and concentrated where possible to ~10 mg/mL. BG505-SOSIP was requested from the Vaccine Research Center at the National Institute of Health (47 (link)).

Sheng Z., Bimela J.S., Katsamba P.S., Patel S.D., Guo Y., Zhao H., Guo Y., Kwong P.D, & Shapiro L. (2022). Structural Basis of Antibody Conformation and Stability Modulation by Framework Somatic Hypermutation. Frontiers in Immunology, 12, 811632.

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Expression and Purification of SARS-CoV-2 Spike Protein

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The SARS-CoV-2 and SARS-CoV S2P spike constructs were produced as previously described [3 (link)]. The proteins were expressed in HEK293 Freestyle cells (Invitrogen) in suspension culture using serum-free media (Invitrogen) and transfected into HEK293 cells using polyethyleneimine (Polysciences). Cell growths were harvested four days after transfection, and the secreted proteins were purified from supernatant by nickel affinity chromatography using Ni-NTA IMAC Sepharose 6 Fast Flow resin (GE Healthcare) followed by size exclusion chromatography on a Superdex 200 column (GE Healthcare) in 10 mM Tris, 150 mM NaCl, pH 7.4. Spike purity was assessed by SDS-PAGE. 2–36 was expressed and purified as previously described [10 (link)]. Fab fragments were produced by digestion of IgGs with immobilized papain at 37 °C for 3 hrs in 50 mM phosphate buffer, 120 mM NaCl, 30 mM cysteine, 1 mM EDTA, pH 7. The resulting Fabs were purified by affinity chromatography on protein A, and purity was assessed by SDS-PAGE.

Wang P., Casner R.G., Nair M.S., Yu J., Guo Y., Wang M., Chan J.F., Cerutti G., Iketani S., Liu L., Sheng Z., Chen Z., Yuen K.Y., Kwong P.D., Huang Y., Shapiro L, & Ho D.D. (2021). A monoclonal antibody that neutralizes SARS-CoV-2 variants, SARS-CoV, and other sarbecoviruses. bioRxiv.

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SARS-CoV-2 RBD Expression and Purification

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The RBD (residues 319 to 541) of the SARS-CoV-2 was expressed and purified as previously described (38). Briefly, protein expression was carried out in suspension cultures of Human Embryonic Kidney (HEK) 293 GnTI-Freestyle cells using serum-free media (Invitrogen) by transient transfection using PEI (Polysciences). Media was harvested four days after transfection and the secreted protein was purified from the supernatant using nickel affinity chromatography using Ni-NTA IMAC Sepharose 6 Fast Flow resin (GE Healthcare) followed by size exclusion chromatography (SEC) on a Superdex 200 column (GE Healthcare) in 20 mM Tris, 150 mM NaCl, pH 8.0.
Fab fragments of 10-40, 10-28, and 11-11 were produced by digestion of IgG antibodies with immobilized Endoproteinase Lys-C (Sigma Aldrich) equilibrated with 25 mM Tris pH 8.5 and 1 mM EDTA for 3 hours. The resulting Fabs were purified from the cleaved Fc domain by affinity chromatography using protein A. Fab purity was analyzed by SDS-PAGE. All Fabs were buffer-exchanged into 20 mM Tris, 150 mM, pH 7.4 prior to cryo-EM or crystallization experiments.

Liu L., Iketani S., Guo Y., Casner R.G., Reddem E.R., Nair M.S., Yu J., Chan J.F., Wang M., Cerutti G., Li Z., Morano N.C., Castagna C.D., Corredor L., Chu H., Yuan S., Poon V.K., Chan C.C., Chen Z., Luo Y., Cunningham M., Chavez A., Yin M.T., Perlin D.S., Tsuji M., Yuen K.Y., Kwong P.D., Sheng Z., Huang Y., Shapiro L, & Ho D.D. (2022). An antibody class with a common CDRH3 motif broadly neutralizes sarbecoviruses. Science Translational Medicine.

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Recombinant SARS-CoV-2 Spike Protein and Fab Production

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The SARS-CoV-2 and SARS-CoV S2P spike constructs were produced as previously described [3 (link)]. The proteins were expressed in HEK293 Freestyle cells (Invitrogen) in suspension culture using serum-free media (Invitrogen) and transfected into HEK293 cells using polyethyleneimine (Polysciences). Cell growths were harvested four days after transfection, and the secreted proteins were purified from supernatant by nickel affinity chromatography using Ni-NTA IMAC Sepharose 6 Fast Flow resin (GE Healthcare) followed by size exclusion chromatography on a Superdex 200 column (GE Healthcare) in 10 mM Tris, 150 mM NaCl, pH 7.4. Spike purity was assessed by SDS-PAGE. 2–36 was expressed and purified as previously described [10 (link)]. Fab fragments were produced by digestion of IgGs with immobilized papain at 37°C for 3 h in 50 mM phosphate buffer, 120 mM NaCl, 30 mM cysteine, 1 mM EDTA, pH 7. The resulting Fabs were purified by affinity chromatography on protein A, and purity was assessed by SDS-PAGE.

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Recombinant SARS-CoV-2 S2P Spike Protein

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SARS-CoV-2 S2P spike was produced as described in (Wrapp et al., 2020 (link)). Protein expression was carried out in Human Embryonic Kidney (HEK) 293 Freestyle cells (Invitrogen) in suspension culture using serum-free media (Invitrogen) by transient transfection using polyethyleneimine (Polysciences). Cell growths were harvested four days after transfection, and the secreted protein was purified from supernatant by nickel affinity chromatography using Ni-NTA IMAC Sepharose 6 Fast Flow resin (GE Healthcare) followed by size exclusion chromatography on a Superdex 200 column (GE Healthcare) in 10 mM Tris, 150 mM NaCl, pH 7.4.

Cerutti G., Rapp M., Guo Y., Bahna F., Bimela J., Reddem E.R., Yu J., Wang P., Liu L., Huang Y., Ho D.D., Kwong P.D., Sheng Z, & Shapiro L. (2021). Structural basis for accommodation of emerging B.1.351 and B.1.1.7 variants by two potent SARS-CoV-2 neutralizing antibodies. Structure (London, England : 1993), 29(7), 655-663.e4.

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