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Ni nitrilotriacetic acid agarose resin

Manufactured by Qiagen
Sourced in Germany

Ni-nitrilotriacetic acid agarose resin is a chromatography media used for the purification of recombinant proteins with a histidine-tag. It utilizes the high-affinity interaction between nickel ions and the histidine residues to capture and isolate the target proteins.

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3 protocols using ni nitrilotriacetic acid agarose resin

1

Purification of GLD-2 Protein from E. coli

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E. coli BL21 transformed with pTrcHisB-GLD-2 were cultured in LB medium containing 100 μg/ml ampicillin. Cells were cultured at 37°C to 0.8 OD at 600 nm, and protein expression was induced at 25°C by the addition of 1 mM isopropyl-β-d-thiogalactopyranoside for 12 h. Cell pellets from a 2-l culture were washed once with phosphate-buffered saline (PBS) and resuspended in 40 ml of binding buffer (50 mM Tris-HCl, pH 8.0, 300 mM NaCl, 10 mM imidazole, 10 mM β-mercaptoethanol, 10% glycerol, 1% Nonidet P-40). Cells were sonicated on ice and centrifuged at 35,000 × g for 15 min. After centrifugation, the supernatant was bound to Ni-nitrilotriacetic acid agarose resin (Qiagen, Hilden, Germany) pre-equilibrated with the binding buffer. Bound proteins were eluted with the binding buffer containing imidazole (50–500 mM). Fractions containing GLD-2 were then dialyzed against buffer A [50 mM Tris-HCl, pH 8.0, 1 mM dithiothreitol (DTT), 50 mM NaCl, 5 mM MgCl2, and 10% glycerol], and aliquots were stored at −80°C.
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2

Recombinant Expression and Purification of SirT6 and SirT7

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Human cDNAs of SirT6 or SirT7 were cloned into the pET-30b expression vector (Novagen, Germany), containing a hexahistidine tag at the C terminus. The constructs were expressed in E. coli BL21 (DE3) grown at 37°C unit optical density at 600 nm of 0.5 and then induced with 1 mM isopropyl-β-d-thiogalactopyranoside for 4 hours. Harvested cells were resuspended in lysis buffer [20 mM Hepes (pH 7.9), 500 mM NaCl, 10% glycerol, 1 mM EDTA (pH 8.0), 0.1% NP-40, 20 mM 2-mercaptoetanol, and 1 mM phenylmethylsulfonyl fluoride (PMSF)] and lysed by sonication. After centrifugation at 12,000g at 4°C for 1 hour, the cleared lysates were loaded onto Ni–nitrilotriacetic acid agarose resin (Qiagen) by gravity flow. After several washes with lysis buffer and BC500 [20 mM tris-HCl (pH 8.0), 500 mM KCl, 10% glycerol, 1 mM EDTA, 1 mM DTT, 0.1% Nonidet P-40, and 0.2 mM PMSF], rSirT7 was eluted using 100 mM imidazole and subsequently dialyzed in BC100. Recombinant proteins were then kept at −80°C.
Preparation of recombinant histones were performed from E. coli Bl21 (DE3) cells as described elsewhere (48 (link)). Histone octamers were concentrated to 3 to 15 mg/ml, adjusted to 50% (v/v) glycerol, and stored at −80°C.
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3

Recombinant Influenza Protein Production

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The wt NA and mutant NA (I223R/H275Y) genes derived from A/H1N1 2009 pandemic influenza virus were amplified by PCR. The PCR products were cloned into the pAcGP67A vector (BD Biosciences) and transfected into the Sf9 insect cell line to produce recombinant proteins. The culture medium was collected, clarified by centrifugation at 110 × g, and subjected to further viral propagation. The cultured Sf9 cells were inoculated with recombinant baculovirus at a multiplicity of infection of 10 and incubated at 27 °C for 96 h. The cells were collected after centrifugation, and the cell pellet was resuspended in cell lysis buffer (50 mM Tris-HCl, pH 8.5, 5 mM 2-mercaptoethanol, 100 mM KCl, 1 mM phenylmethylsulfonyl fluoride), sonicated, and centrifuged at 16,000 × g for 15 min at 4 °C. After the cell lysate was sonicated to reduce its viscosity, the cell debris was removed by centrifugation for 1 h at 16,000 × g. The soluble protein from the cell supernatant was applied to Ni-nitrilotriacetic acid agarose resin (Qiagen), washed, and eluted with buffer (50 mM Tris-HCl, 0.5 M NaCl, 0.5 M imidazole, pH 8.0). The purified proteins were dialyzed against phosphate-buffered saline (PBS) and further purified by Q-Sepharose anion-exchange chromatography (GE Healthcare).
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