Ni2 nitrilotriacetic acid column

Manufactured by Qiagen

The Ni2+-nitrilotriacetic acid column is a laboratory equipment used for the purification and isolation of recombinant proteins. The column contains nickel-charged nitrilotriacetic acid (Ni2+-NTA) resin, which selectively binds to proteins with a histidine tag, allowing for their effective separation from other cellular components.

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Lab products found in correlation

Isopropyl β d thiogalactopyranoside iptg, by Merck Group (1 mentions) One shot cell disruptor, by Constant Systems (1 mentions) Bl21 de3 cells, by Thermo Fisher Scientific (1 mentions)

Spelling variants of this product

Ni2+-nitrilotriacetic acid Sepharose affinity column (4 citations) Ni2+-nitrilotriacetic acid Sepharose Superflow column (2 citations)

4 protocols using ni2 nitrilotriacetic acid column

Recombinant GFP-Dnmt1 Protein Production

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To produce recombinant GFP-Dnmt1 proteins, PCR was performed to obtain the GFP-Dnmt1s^c− part using the pEGFP-GFP-Dnmt1s^c− plasmid as a template under the condition of 20 cycles of 94°C/30 s, 46°C/45 s and 72°C/2.5 m (MJ gradient cycler). The primers used were 5′-GCGATATCATGGTGAGCAAGG-3′ and 5′-GCGATATCCTAGATCCGGTG-3′. The PCR product was cloned into EcoRV (NEB) site of pET32 vector (Novagen) to make pET-32a(+)-GFP-Dnmt1s^c−. GFP-Dnmt1s^c− recombinant protein was produced after transformation into E. coli BL21 (DE3) strains and induction using 0.25 mM isopropyl-β-d-thiogalactopyranoside (IPTG; Sigma-Aldrich) for 2 or 4 h. The recombinant protein was purified using Ni²⁺-nitrilotriacetic acid column (Qiagen) and concentrated by Centricon (Amicon) according to the manufacturer’s instructions. For microinjection, the GFP-Dnmt1s^c− recombinant protein at ∼1 μg/μl concentration was injected into the cytoplasm of a single blastomere of two-cell embryo. Three hours later, GFP was observed on the fluorescence microscope as mentioned earlier.

Min B., Park J.S., Jeong Y.S., Jeon K, & Kang Y.K. (2020). Dnmt1 binds and represses genomic retroelements via DNA methylation in mouse early embryos. Nucleic Acids Research, 48(15), 8431-8444.

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Purification of Mph Enzyme from E. coli

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Mphs were purified as described previously for MphI^{36 (link)}. E. coli BL21(DE3) was cultured with shaking at 37 °C in 1 L of LB-Lennox until an OD₆₀₀ of 0.5–0.6, chilled in an ice bath for 20 min, and protein overexpression induced by the addition of 1 mM IPTG and incubation at 17 °C for 16 h. Cell mass was harvested by centrifuging at 10,000 × g for 20 min, washed with cold saline, and resuspended in 20 mL buffer A (50 mM HEPES pH 7.5, 150 mM NaCl, 5% glycerol, 10 mM imidazole). Cell lysis was performed using a One-shot Cell Disruptor (Constant Systems Limited) at 20,000 psi, and cell debris removed after adding 15 mL buffer A, 5 mg bovine bovine pancreas DNase, and 2.5 mg of bovine pancreas RNase by centrifugation at 50,000 × g for 45 min. Proteins were purified using 1 mL Ni²⁺-nitrilotriacetic acid column (Qiagen) and a linear gradient of 95% buffer A to 100% buffer B (buffer A with 250 mM imidazole) over 20 column volumes. Fractions with pure Mph were pooled and desalted into 50 mM HEPES pH 7.5 using a PD-10 desalting column (GE Scientific). Pure enzyme stocks were stored at 4 °C.

Pawlowski A.C., Stogios P.J., Koteva K., Skarina T., Evdokimova E., Savchenko A, & Wright G.D. (2018). The evolution of substrate discrimination in macrolide antibiotic resistance enzymes. Nature Communications, 9, 112.

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Purification of ArsV Protein from E. coli

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E. coli BL21(DE3) cells (Life Technologies) bearing plasmid pET29a-ArsV were grown in LB medium containing 50 μg ml⁻¹ kanamycin with shaking at 37°C. Cells at an A_600nm of 0.6 were induced by addition of 0.3 mM IPTG and cultured for an additional 4 h. The cells were harvested and suspended in 5 ml per gram of wet cells in buffer A (50 mM 4-morpholinepropanesulfonic acid, 20 mM imidazole, 0.5 M NaCl, 10 mM 2-mercaptoethanol and 20% glycerol (vol/vol), pH 7.5). The cells were broken by a single passage through a French pressure cell at 20000 psi and immediately treated with diisopropyl fluorophosphate (2.5 μl per gram wet cell). Membranes and unbroken cells were removed by centrifugation at 150 000 g for 1 h, and the supernatant solution was loaded onto a Ni²⁺-nitrilotriacetic acid column (Qiagen, Valencia, CA) at a flow rate of 0.5 ml min⁻¹. The column was washed with more than 25 column volumes of buffer A. ArsV was eluted with buffer A containing 0.2 M imidazole, and the purity was analysed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (Laemmli, 1970 (link)). Protein concentrations were estimated from the A_280nm (ε = 38,850 M⁻¹ cm⁻¹; Gill and von Hippel, 1989 (link)). ArsV-containing fractions were divided into small portions, rapidly frozen and stored at −80°C until use.

Chen J., Zhang J., Wu Y.F., Zhao F.J, & Rosen B.P. (2021). ArsV and ArsW provide synergistic resistance to the antibiotic methylarsenite. Environmental microbiology, 23(12), 7550-7562.

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Purification of PpArsH Protein from E. coli

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E. coli TOP10 cells (Life Technologies) bearing PpArsH in vector plasmid pBAD/myc-HisA were grown in Luria-Bertani medium containing 100 µg/ml ampicillin with shaking at 37 °C. At an A_{600 nm} of 0.6, L-arabinose was added as an inducer to a final concentration of 0.2% (w/v), and the culture was grown for an additional 4 h at 37 °C. The cells were harvested and suspended in 5 ml per gram of wet cells in buffer A (50 mM 4-morpholinepropanesulfonic acid, 20 mM imidazole, 0.5 M NaCl, 10 mM 2-mercaptoethanol and 20% glycerol (vol/vol), pH 7.5). The cells were broken by a single passage through a French pressure cell at 20,000 psi, and immediately treated with diisopropyl fluorophosphate (2.5 µl per gram wet cell). Membranes and unbroken cells were removed by centrifugation at 150,000 × g for 1 h, and the supernatant solution was loaded onto a Ni²⁺-nitrilotriacetic acid column (Qiagen, Valencia, CA) at a flow rate of 0.5 ml/min. The column was washed with more than 25 column volumes of buffer A. PpArsH was eluted with buffer A containing 0.2 M imidazole, and the purity was analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE). Protein concentrations were estimated from A_{280 nm} (ε = 28,120 M⁻¹ cm⁻¹). PpArsH-containing fractions were divided into small portions, rapidly frozen, and stored at −80 °C until use.

Chen J., Bhattacharjee H, & Rosen B.P. (2015). ArsH is an organoarsenical oxidase that confers resistance to trivalent forms of the herbicide MSMA and the poultry growth promoter roxarsone. Molecular microbiology, 96(5), 1042-1052.

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