Ni nta

Manufactured by GenScript

Sourced in China

Ni-NTA is a nickel-nitrilotriacetic acid (Ni-NTA) resin used for the purification of recombinant proteins with a histidine tag. It binds to the histidine tag, allowing the target protein to be isolated from the sample mixture.

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Lab products found in correlation

Expi293f, by Thermo Fisher Scientific (1 mentions) Strep tactin xt, by IBA Lifesciences (1 mentions) Superdex 200 increase 10 300, by GE Healthcare (1 mentions) Pei max, by Polysciences (1 mentions) Superdex 75 increase 10 300 gl, by GE Healthcare (1 mentions) Starion fla 9000 instrument, by Fujifilm (1 mentions)

4 protocols using ni nta

Purification of SARS-CoV-2 RBD Proteins

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The recombinant proteins were overexpressed in E. coli BL21 (DE3). After overnight induction by 0.1 mM isopropyl β-D-thiogalactoside (IPTG) at 16 °C in LB medium, cells were harvested and suspended in buffer (40 mM Tris-HCl, pH 8.0; 500 mM NaCl; 2mM phenylmethylsulfonyl fluoride). After cell lysis and centrifugation, the recombinant proteins were purified by Ni-NTA (GenScript) affinity chromatography. Ion exchange columns and gel-filtration chromatography (SD200, Superdex200 Increase 10/300, GE Healthcare) were used for further purification.
SARS-CoV-2 RBD wild-type proteins (with an N-terminal signal peptide and a C-terminal TwinStrep tag or 8 × His tag or TwinStrep-KKETPV tag) were expressed in Expi293F (Thermo Fisher Scientific) cells at 37 °C in a humidified 5% CO₂ incubator rotating at 120 r.p.m. Transfections were performed using PEI MAX (Polysciences) at a DNA:PEI ratio of 1:3. After cultivation for 96 h the supernatants were collected and purified by Ni-NTA (GenScript) affinity chromatography or Strep-Tactin® XT (IBA Lifesciences) according to the manufacturer's protocol. Ion exchange columns and gel-filtration chromatography (SD200, Superdex200 Increase 10/300, GE Healthcare) were used for further purification. Recombinant hACE2 protein was produced as described previously with some modifications [22] (link).

Pei G., Xu W., Lan J., Wang X, & Li P. (2022). CEBIT screening for inhibitors of the interaction between SARS-CoV-2 spike and ACE2. Fundamental Research, 2(4), 562-569.

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Expression and Purification of PARC6 and PDV1 Proteins

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Various segments of PARC6C and PDV1 were amplified by PCR and cloned into the pET28a vector. The protein was expressed and purified as previously described (37 (link)). Briefly, the proteins were expressed in Escherichia coli strain BL21 (DE3), and the bacteria were cultured at 18 °C overnight for the protein expression. The cell lysate was centrifuged to remove cell debris. Then, the supernatant was applied onto a self-packaged Ni-affinity column (2 mL Ni-NTA, Genscript). The fusion protein was eluted with elution buffer (50 mM Tris pH 8.0, 300 mM NaCl, 300 mM imidazole). The eluant of PARC6^640–819 was concentrated and further purified using a Superdex-75 increase 10/300 GL (GE Healthcare) column equilibrated with a buffer containing 10 mM Tris-HCl pH 8.0 and 200 mM NaCl. The eluant of PARC6^685–819–PDV1^263–272 was concentrated and further purified using a Superdex-75 increase 10/300 GL (GE Healthcare) column equilibrated with a buffer containing 10 mM Tris-HCl pH 8.0, 200 mM NaCl, and 5 mM DTT. The purified protein was analyzed by SDS-PAGE. Fractions containing the target protein were pooled and concentrated.

Sun Q., Cao X., Liu Z., An C., Hu J., Wang Y., Qiao M., Gao T., Cheng W., Zhang Y., Feng Y, & Gao H. (2023). Structural and functional insights into the chloroplast division site regulators PARC6 and PDV1 in the intermembrane space. Proceedings of the National Academy of Sciences of the United States of America, 120(5), e2215575120.

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Recombinant SARS-CoV-2 Protein Purification

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The coding sequences of SARS-CoV nsp10, 16; SARS-CoV-2 nsp10, 14, 16; and mutants were cloned into a pET32a vector with the His tag. E. coli BL21 (DE3) cells were transformed with the respective plasmid and the recombinant protein was induced with 0.4 mM isopropyl β-d-thiogalactopyranoside (IPTG) at 16 °C for 12–16 h. The cells were harvested by centrifugation, and the pellets were resuspended in lysis buffer (50 mM Tris–HCl, pH 8.0, 300 mM NaCl, 10% glycerol, and 5 mM MgCl₂). The cells were then disrupted by a high-pressure cracker (UH-24, Union-biotech, Shanghai, China), and cell debris was removed by centrifugation. pET32a-His6-nsp10, 14, and 16 were purified with nickel-nitrilotriacetic acid (Ni-NTA, Shanghai, China) resin (GenScript, Piscataway, NJ, USA) as described previously [45 (link),46 (link)].

Wu K., Guo Y., Xu T., Huang W., Guo D., Cao L, & Lei J. (2024). Structure-Based Virtual Screening for Methyltransferase Inhibitors of SARS-CoV-2 nsp14 and nsp16. Molecules, 29(10), 2312.

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Protein Expression and Purification of OsGRF8 WRC Domain

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For protein expression and purification, the DNA binding domain of OsGRF8 (WRC) was cloned into the pET44b vector and transformed into Escherichia coli strain BL21 to produce His-tagged fusion protein. His-WRC fusion protein was induced by adding 0.5 mM isopropyl-d-1-thiogalactopyranoside to the culture medium and incubating the cells for 14 h at 20°C and purified using Ni-NTA (nitrilotriacetic acid) agarose (GenScript) according to the manufacturer’s instructions. The EMSAP1, EMSAP2, EMSAP1t, and EMSAP2t DNA probes from the OsMIR408 promoter were synthesized and cy5 labeled. The DNA probes and proteins were co-incubated in the reaction buffer, purified and incubated with the Cy5-labeled probe at 25°C for 20 min in EMSA buffer (25 mM HEPES (pH 7.5), 40 mM potassium chloride, 3 mM dithiothreitol, 10% (v/v) glycerol, 0.1 mM EDTA, 0.5 mg/ml bovine serum albumin, 0.5 mg/ml poly-glutamate). After incubation, the reaction mixture was electrophoresed on a 6% native polyacrylamide gel, and then labeled DNA was detected using a Starion FLA-9000 instrument (Fujifilm, Tokyo, Japan).

Yang X., Zhao X., Dai Z., Ma F., Miao X, & Shi Z. (2021). OsmiR396/growth regulating factor modulate rice grain size through direct regulation of embryo-specific miR408. Plant Physiology, 186(1), 519-533.

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