Ni affinity column

Manufactured by GE Healthcare

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The Ni affinity column is a laboratory equipment used for the purification of recombinant proteins containing a histidine tag. It utilizes the high-affinity interaction between nickel ions and the histidine residues to capture and separate the target protein from other components in a sample.

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Lab products found in correlation

Superdex 200, by GE Healthcare (2 mentions) Superdex 75, by GE Healthcare (2 mentions) Quikchange 2 mutagenesis kit, by Agilent Technologies (1 mentions) Fastpfu dna polymerase, by Transgene (1 mentions) Superdex 75 column, by GE Healthcare (1 mentions) Glutathione sepharose 4b beads, by GE Healthcare (1 mentions) Anti flag, by Thermo Fisher Scientific (1 mentions)

Spelling variants of this product

Ni-NTA affinity column (22 citations) Ni-NTA affinity chromatography column (9 citations) Ni-affinity chromatography column (3 citations) HiTrap chelating HP Ni-affinity columns (2 citations) HiTrap columns for Ni-NTA affinity chromatography (2 citations) HIS GraviTrap Ni-nitrilotriacetic acid (Ni-NTA) affinity columns (2 citations) HisTrap Ni-affinity column (2 citations) Immobilized metal affinity Ni+ charged sepharose column (2 citations) 1 ml HisTrap Ni-Sepharose affinity column (2 citations) Ni-nitrilotriacetic acid affinity column (2 citations)

5 protocols using ni affinity column

Purification of Ssa1 and Mutants

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Full-length Ssa1 (residues 2-642), truncated Ssa1 (residues 382-554) and corresponding mutations were purified as previously described (24) . Briefly, full-length Ssa1 and mutations were firstly purified on a Ni affinity column (GE Healthcare) in 50 mM Tris buffer (pH 7.5), 300 mM NaCl and 3 mM β-mercaptoethanol. Following Ulp1 cleavage to remove the N-terminal His-Smt3 tag and a second Ni affinity column purification, the flow through including tag-free protein was further purified by gel filtration chromatography (Superdex 200, GE Healthcare) in 50 mM Tris buffer (pH 7.5), 100 mM KCl and 5 mM MgCl2. The truncation mutants were similarly purified with some modifications to buffers.
For Ni affinity purification, 50 mM Tris buffer (pH 8.0) and 200 mM NaCl was used. Gel filtration chromatography was performed in 50 mM Na-phosphate buffer (pH 7.0) with 50 mM NaCl (Superdex 75, GE Healthcare).

Xu L., Zhang H., Cuskelly D.D., Doyle S., Perrett S, & Jones G.W. (2021). Mutational analysis of the Hsp70 substrate-binding domain: Correlating molecular-level changes with in vivo function. Molecular microbiology, 115(6).

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Overexpression and Purification of TmoX1062

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The full-length tmoX₁₀₆₂ gene was amplified from the genomic DNA of HTCC1062 by PCR using FastPfu DNA polymerase (TransGen Biotech, China), and was subcloned into the NdeI/XhoI restriction sites of the pET22b (Novagen, America) vector with a C-terminal His-tag. All of the point mutations in tmoX₁₀₆₂ were performed with the QuikChange^® mutagenesis kit II (Agilent, America). The wild-type (WT) TmoX₁₀₆₂ protein and all of the mutants were expressed in E. coli strain BL21(DE3). The recombinant E. coli strains were cultured at 37°C in LB medium to an OD₆₀₀ of 0.8–1.0 and then incubated at 16°C for 16 h with 0.5 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) as an inducer for recombinant protein expression. The recombinant proteins were purified first with Ni-affinity column (GE Healthcare, America), and then with gel filtration on a Superdex-75 column (GE Healthcare, America) eluted with the buffer containing 10 mM Tris–HCl (pH 8.0) and 100 mM NaCl. Approximately 2 mg recombinant TmoX₁₀₆₂ protein was obtained from 1 liter of culture.

Gao C., Zhang N., He X.Y., Wang N., Zhang X.Y., Wang P., Chen X.L., Zhang Y.Z., Ding J.M, & Li C.Y. (2022). Characterization of the Trimethylamine N-Oxide Transporter From Pelagibacter Strain HTCC1062 Reveals Its Oligotrophic Niche Adaption. Frontiers in Microbiology, 13, 838608.

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Recombinant Protein Expression in E. coli

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The CAD coding region was amplified from Z58 and bm1-E plants and then sub-cloned into pET32a vector. The recombinant vectors were transferred into BL21 Escherichia coli cells. Cells were cultured at 37°C in Luria-Bertani medium. Isopropyl β-D-thiogalactopyranoside (IPTG) was added to a final concentration of 0.3 mM at mid-log phase (A600 = 0.4∼0.6). Cells were continued to be incubated at 18°C for 16 h and harvested by centrifugation at 5,000 × g for 5 min. Soluble proteins were extracted by sonication and the recombinant proteins were purified with Ni affinity column (GE Healthcare) for further enzyme activity (Youn et al., 2006 (link)).

Xiong W., Li Y., Wu Z., Ma L., Liu Y., Qin L., Liu J., Hu Z., Guo S., Sun J., Yang G., Chai M., Zhang C., Lu X, & Fu C. (2020). Characterization of Two New brown midrib1 Mutations From an EMS-Mutagenic Maize Population for Lignocellulosic Biomass Utilization. Frontiers in Plant Science, 11, 594798.

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Purification of Full-length and Truncated Ssa1

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Full-length Ssa1 (residues 2–642), truncated Ssa1 (residues 382–554) [51 ] and corresponding mutations were constructed in the pET28a-Smt3 vector containing 6× His and Smt3 tags [52 (link)]. The plasmid was transformed into BL21-CodonPlus (DE3)-RIL competent cells and induced by IPTG in 2YT medium. For NMR, M9 minimal medium containing ¹⁵N–NH₄Cl or ¹⁵N–NH₄Cl/¹³C–glucose was used. Full-length Ssa1 and mutations were first purified on an Ni affinity column (GE Healthcare) in 50 mM Tris buffer (pH 7.5), 300 mM NaCl, and 3 mM β-mercaptoethanol. Protein product was then incubated with Ulp1 followed by a second Ni affinity column purification step to remove the 6× His-Smt3 tag, Ulp1, and un-cleaved protein. The flow through was collected and further purified by gel filtration chromatography (Superdex 200, GE Healthcare) in 50 mM Tris buffer (pH 7.5), 100 mM KCl, and 5 mM MgCl₂. The truncation mutants were similarly purified with some modifications to buffers. For Ni affinity purification, 50 mM Tris buffer (pH 8.0) and 200 mM NaCl were used. Gel filtration chromatography was performed in 50 mM Na–phosphate buffer (pH 7.0) with 50 mM NaCl (Superdex 75, GE Healthcare).

Xu L., Gong W., Cusack S.A., Wu H., Loovers H.M., Zhang H., Perrett S, & Jones G.W. (2017). The β6/β7 region of the Hsp70 substrate-binding domain mediates heat-shock response and prion propagation. Cellular and Molecular Life Sciences, 75(8), 1445-1459.

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Purification and Interaction of SeASF1 and SeGtr1

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The Seasf1 was cloned into the pET28a vector after adding a 1×FLAG tag to the 5′-terminal of Seasf1 to make the Flag-SeASF1-His fusion protein. The Segtr1 was cloned into the pGEX-6P-1 vector to make the GST-SeGtr1 fusion protein (Table 2). Flag-SeASF1-His, GST-SeGtr1, pET28a or pGEX-6P-1 was expressed in BL21 strain of E. coli, and were then affinity purified with a Ni-affinity column (GE) or GST-affinity column (glutathione sepharose^TM 4B beads GE Healthcare, Little Chalfont, Buckinghamshire, UK). For glutathione S-transferase (GST) pull-down in vitro, GST-SeGtr1 and Flag-SeASF1-28a were expressed in E. coli strain BL21 (DE3). Total proteins of GST-SeGtr1 and Flag-SeASF1-His were then incubated with 4000 μL of glutathione sepharose^TM 4B beads at 4 °C for 2 h. The supernatant was removed, and the beads was washed by GST-lysis buffer three times. Finally, the beads were eluted by GST-elution buffer. Pull-down of GST-SeGtr1 with Flag-SeASF1-His was detected using an anti-Flag (Invitrogen, Waltham, MA, USA). Each experiment was repeated at least three times.

Wang S., Song C., Zhao L., Xu W., Li Z., Liu X, & Zhang X. (2022). GTP Binding Protein Gtr1 Cooperating with ASF1 Regulates Asexual Development in Stemphylium eturmiunum. International Journal of Molecular Sciences, 23(15), 8355.

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