Hitrap nickel column

Manufactured by GE Healthcare

Sourced in France

The HiTrap nickel column is a pre-packed chromatography column designed for the purification of His-tagged recombinant proteins. It is filled with agarose beads coated with nickel ions, which bind to the histidine residues present on the target proteins. The column can be used in a variety of protein purification workflows, including fast protein liquid chromatography (FPLC) systems.

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Lab products found in correlation

Pierce protein a igg plus orientation kit, by Thermo Fisher Scientific (2 mentions) Freestyle 293t, by Thermo Fisher Scientific (2 mentions) Gnt1 293t mammalian expression system, by Thermo Fisher Scientific (2 mentions) Pmal c5e expression vector, by New England Biolabs (2 mentions) Bovine thrombin, by Merck Group (1 mentions) Superdex 200 column, by GE Healthcare (1 mentions) Imidazole, by Euromedex (1 mentions)

Spelling variants of this product

Nickel column (27 citations) HiTrap columns (11 citations) HiTrap nickel affinity column (2 citations) HiTrap™ HP nickel column (2 citations) HiTrap nickel-chelating HP column (1 citations)

6 protocols using hitrap nickel column

HK Purification and Glucose Assay

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Human HK‐I and HK‐II were cloned into plasmid pET‐His DNA and expressed in Escherichia coli BL21 upon IPTG induction. HK was purified from lysed cells using a HiTrap nickel column (GE Healthcare). Following dialysis, the imidazole‐eluted protein was further purified using a Blue HiTrap column (GE Healthcare). HK was eluted using 10 mm glucose, 20 mm Tris/HCl (pH 8.5), 20% glycerol, and 1.5 mm glucose‐6‐phosphate. Fractions were analyzed by SDS/PAGE and tested for HK activity. The purified active fractions were combined and stored in aliquots at −80 °C.
Purified HK‐I (13 μg·mL⁻¹) was preincubated with Tf‐D‐LP4 or R‐Tf‐D‐LP4 peptides in reaction buffer comprising 20 mm HEPES/KOH (pH 7.8), 10 mm glucose, 4 mm MgCl₂, and 0.6 mm NADP and assayed for HK activity. Change in absorbance at 340 nm (NADH production) was spectrophotometrically measured following addition of 1 mm ATP and 0.05 unit·mL⁻¹ glucose‐6‐phosphate dehydrogenase.

Shteinfer‐Kuzmine A., Amsalem Z., Arif T., Zooravlov A, & Shoshan‐Barmatz V. (2018). Selective induction of cancer cell death by VDAC1‐based peptides and their potential use in cancer therapy. Molecular Oncology, 12(7), 1077-1103.

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Functional and Structural Analysis of IDs

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IDs were expressed by transfection using the FreeStyle 293T and GnT1⁻ 293T Mammalian Expression System (Invitrogen) for functional and structural analysis, respectively, and according to the protocol provided by the manufacturer. IDs were purified using an N5-i5 IgG affinity column. N5-i5 IgG was chemically crosslinked to protein A resin using the Pierce Protein A IgG Plus orientation kit (Thermo Fisher Scientific). Protein bound to the N5-i5 IgG affinity column was eluted with 0.1 M glycine (pH 3.0) and immediately diluted 10:1 with 1 M Tris-HCl (pH 8.5).
ID2 for co-crystallization studies was grown in the Origami (DE) Escherichia coli strain (Novagen). ID2 sequence was cloned into the pMal-c5e expression vector (New England Biolabs) with an N-terminal maltose-binding protein (MBP)-thioredoxin tag followed by a six-histidine tag and a thrombin cleavage sequence. The cell lysate (after 2–3 min of sonication and centrifugation at 12,000 × g for 30 min) was loaded onto a HiTrap nickel column (GE Healthcare). MBP-thioredoxin-ID2 was eluted with 25 mM Tris-HCl (pH 8.0) and 500 mM imidazole (pH 8.0). The MBP-thioredoxin tag was removed by digestion overnight at 4°C with agarose-linked bovine thrombin (Sigma) and passing the lysate over an amylose column. Flow-through fractions were concentrated and purified further using an N5-i5 IgG affinity column as described above.

Tolbert W.D., Gohain N., Veillette M., Chapleau J.P., Orlandi C., Visciano M.L., Ebadi M., DeVico A.L., Fouts T.R., Finzi A., Lewis G.K, & Pazgier M. (2016). Paring Down HIV Env: Design and Crystal Structure of a Stabilized Inner Domain of HIV-1 gp120 Displaying a Major ADCC Target of the A32 Region. Structure(London, England:1993), 24(5), 697-709.

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Protein Expression, Purification, and Crosslinking

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IDs were expressed by transfection using the FreeStyle^™ 293T and GnT1⁻ 293T Mammalian Expression System (Invitrogen, Carlsbad, CA) for functional and structural analysis, respectively, and according to the protocol provided by the manufacturer. IDs were purified using an N5-i5 IgG affinity column. N5-i5 IgG was chemically crosslinked to protein A resin using the Pierce protein A IgG plus orientation kit (Thermo Fisher). Protein bound to the N5-i5 IgG affinity column was eluted with 0.1 M glycine, pH 3.0 and immediately diluted 10:1 with 1 M Tris-HCl pH 8.5.
ID2 for co-crystallization studies was grown in the Origami (DE) E. coli strain (Novagen). ID2 sequence was cloned into the pMal-c5e expression vector (New England Biolabs) with an N-terminal maltose binding protein (MBP)-thioredoxin tag followed by a six-histidine tag and a thrombin cleavage sequence. The cell lysate (after 2 to 3 minutes of sonication and centrifugation at 12,000 × g for 30 minutes) was loaded onto a HiTrap nickel column (GE Healthcare). MBP-thioredoxin-ID2 was eluted with 25 mM Tris-HCl pH 8.0, 500 mM imidazole pH 8.0. The MBP-thioredoxin tag was removed by digestion overnight at 4° C with agarose linked bovine thrombin (Sigma) and passing the lysate over an amylose column. Flow-through fractions were concentrated and purified further using an N5-i5 IgG affinity column as described above.

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Recombinant Human IL-6 Protein Purification

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The rhIL-6 gene (Uniprot P05231) was synthesized by GeneArt, Life Technologies and cloned into a CMV promoter-driven expression vector bearing EBNA-1 and oriP with a His₁₀ tag. rhIL-6 was expressed in HEK293F cells and purified on a HiTrap nickel column (GE Healthcare Life Sciences). Protein was eluted with 250 mm imidazole and dialyzed into PBS. Aggregates were removed using a Superdex 200 column (GE Healthcare). Carrier-free rhIL-6 for use in in vivo experiments was purchased from R&D Systems (Minneapolis, MN).

Kasturirangan S., Rainey G.J., Xu L., Wang X., Portnoff A., Chen T., Fazenbaker C., Zhong H., Bee J., Zeng Z., Jenne C., Wu H, & Gao C. (2017). Targeted Fcγ Receptor (FcγR)-mediated Clearance by a Biparatopic Bispecific Antibody. The Journal of Biological Chemistry, 292(10), 4361-4370.

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Purification of SeMet-Derivatized Protein

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Harvested cells of either native or selenomethionine-derivatized (SeMet) SmhA were defrosted, resuspended in lysis buffer (50 mM Tris pH 8.0) and lysed by sonication (3 × 20 s bursts at 16 µm amplitude). Insoluble material was removed by centrifugation at 40 000g for 15 min. The supernatant was applied onto a 5 ml nickel HiTrap column (GE Healthcare) in binding buffer (50 mM Tris pH 8.0, 0.5 M NaCl). The protein was eluted with a linear gradient of 0–1 M imidazole in binding buffer and fractions containing protein were pooled, concentrated and buffer-exchanged into 50 mM Tris pH 8.0, 10 mM NaCl for crystallization; the purification was analysed by SDS–PAGE. Macromolecule-production information is summarized in Table 1 ▸.

Churchill-Angus A.M., Sedelnikova S.E., Schofield T.H, & Baker P.J. (2020). The A component (SmhA) of a tripartite pore-forming toxin from Serratia marcescens: expression, purification and crystallographic analysis. Acta Crystallographica. Section F, Structural Biology Communications, 76(Pt 12), 577-582.

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Recombinant Mouse Creatine Kinase B Expression

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The N-terminal His-tagged cDNA coding region for the mouse Ckb (residues 1-318, accession number NM_021273; ref. MR205953, Origene, Herford, Germany) was cloned into the vector pET15b (CIGEx, CEA, Fontenay aux Roses, France). The N-terminal hexahistidine-tagged fusion protein was expressed in Escherichia coli BL21 (DE3). Cells were grown in LB medium (Euromedex) at 37°C until an A₆₀₀ of 0.4 and subsequently induced for 3 h at 37°C with 0.1 mM isopropyl 1-thio-D-galactopyranoside (Euromedex). Harvested bacterial pellets were resuspended in buffer containing isopropyl thio-β-d-galactoside (IPTG; Euromedex). The cell pellets were resuspended in binding buffer (10 mM Tris-HCl, 300 mM NaCl, 10% glycerol, 2 mM CHAPS and 10 mM imidazole, pH 8.0; Euromedex), lysed by sonication on ice (8 × 30 s, 90 W) and then clarified by centrifugation (20,000× g, 50 min, 4°C). The cleared supernatant was loaded on a nickel-Hitrap column (GE Healthcare; Aulnay Sous Bois, France). The protein was eluted using 50 and 250 mM imidazole (10 mM Tris-HCl, 300 mM NaCl, pH 8.0; Euromedex). The protein was then dialyzed 12 h at 4°C against a buffer containing 10 mM Tris, and 150 mM NaCl (pH 8). The purity and homogeneity of the protein were assessed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE; see below).

Weinsanto I., Mouheiche J., Laux-Biehlmann A., Delalande F., Marquette A., Chavant V., Gabel F., Cianferani S., Charlet A., Parat M.O, & Goumon Y. (2018). Morphine Binds Creatine Kinase B and Inhibits Its Activity. Frontiers in Cellular Neuroscience, 12, 464.

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