Ni nta

Manufactured by GoldBio

Ni-NTA (Nickel-Nitrilotriacetic Acid) is a chromatography resin used for the purification of proteins with a histidine-tag. It provides a simple and efficient method for the purification of recombinant proteins. The Ni-NTA resin binds to the histidine-tag on the target protein, allowing it to be separated from other cellular components during the purification process.

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Lab products found in correlation

Hifi dna assembly mix, by New England Biolabs (1 mentions)

Close spelling variants of this product

Ni-NTA chromatography Ni-NTA resin Ni–NTA agarose

3 protocols using ni nta

AmaD Protein Expression and Purification

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The amaD gene was PCR-amplified from P. putida and was cloned in pET22b+ vector void of the pelB sequence using Hi-Fi DNA Assembly mix (New England Biolabs). The pET22b+_amaD vector was electroporated into E. coli Rosetta II (Invitrogen) and selected on carbenicillin. The bacteria were grown in LB to an OD₆₀₀ of 0.6, and AmaD protein expression was induced by the addition of 0.5 mM isopropyl-β-d-thiogalactopyranoside for 3 hours at 30°C. Cells were harvested and lysed with two rounds of a cell disruptor using 35 k.p.s.i. (Constant Systems Ltd., Kennesaw, GA). Cell lysates were clarified at 10,000 rpm for 10 min and then were passed over a nickel-nitrilotriacetic acid-agarose column Ni-NTA (GoldBio, St. Louis, MO). Protein was eluted with 300 mM imidazole, buffer-exchanged using Sephadex G-25 PD10 column, and stored in 50 mM tris-HCl (pH 8.0) and 150 mM NaCl at −80°C.
P. aeruginosa Tse1 protein was obtained as described (18 (link)). LdtA from V. cholerae was obtained as described (22 (link)).

Le N.H., Peters K., Espaillat A., Sheldon J.R., Gray J., Di Venanzio G., Lopez J., Djahanschiri B., Mueller E.A., Hennon S.W., Levin P.A., Ebersberger I., Skaar E.P., Cava F., Vollmer W, & Feldman M.F. (2020). Peptidoglycan editing provides immunity to Acinetobacter baumannii during bacterial warfare. Science Advances, 6(30), eabb5614.

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Multimerization and Fusion of Nanobodies

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Nanobodies were multimerized to dimeric or trimeric forms with (G4S) linkers. The dimeric forms were generated using 6 × (G4S) linker to connect two monomeric VHHs in tandem N terminus to C terminus orientation. Trimers were generated using two 3 × (G4S) linkers between monomeric nanobody units. The multimers were cloned into pET-26b vector adding a C-terminal 6xHis tag. Nanobodies were purified from the periplasmic fraction by Ni-NTA chromatography (Gold Biotechnology) and dialyzed against PBS to remove imidazole.
To generate VHH-IgA fusionbodies, monomeric nanobody sequences were cloned into a pcDNA 3.1 vector containing heavy chain constant region of IgA1 or IgA2 chains without the CH1 domain. Each vector was transformed in NEB5 competent cells, and sequences were verified ahead of transient transfection.

Amcheslavsky A., Wallace A.L., Ejemel M., Li Q., McMahon C.T., Stoppato M., Giuntini S., Schiller Z.A., Pondish J.R., Toomey J.R., Schneider R.M., Meisinger J., Heukers R., Kruse A.C., Barry E.M., Pierce B.G., Klempner M.S., Cavacini L.A, & Wang Y. (2021). Anti-CfaE nanobodies provide broad cross-protection against major pathogenic enterotoxigenic Escherichia coli strains, with implications for vaccine design. Scientific Reports, 11, 2751.

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Protein Expression and Purification

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Protein expression and purification was performed as described previously.4 Briefly, genes were expressed in BL21(DE3) cells at 16 °C for 24 h and 72 h, with the exception of pgiMA1, pgiMA1_mut, and gymMA1, which were expressed in Rosetta(DE3) cells. Cells were harvested and lysed using either French Press or sonication. Recombinant proteins were purified via nickelchelate chromatography based on manufacturers' recommendations (Ni-NTA, Gold Biotechnology). For CmiMA, CmaMA, LedMA, MroMA1, and SveMA, imidazole was removed and protein was concentrated using Amicon Ultra filters (30-kDa MWCO) and loaded onto a pre-equilibrated Superdex-200 Increase size exclusion column. Dimeric protein was collected and concentrated to 4.0 mg/mL, determined by Pierce BCA protein assay kit. For GjuMA and PocMA, a HiLoad 16/600 Superdex 200 pg size exclusion column was used.

Quijano M.R., Zach C., Miller F.S., Lee A.R., Imani A.S., Künzler M, & Freeman M.F. (2019). Distinct Autocatalytic α- N-Methylating Precursors Expand the Borosin RiPP Family of Peptide Natural Products. Journal of the American Chemical Society, 141(24).

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