Nickel affinity chromatography column

Manufactured by GE Healthcare

Sourced in United States

The Nickel-affinity chromatography column is a laboratory equipment used for the purification and isolation of proteins that contain a histidine tag or have a natural affinity for nickel ions. The column is filled with a resin that binds to these proteins, allowing them to be separated from other components in a sample. The core function of this column is to facilitate the capture, concentration, and purification of target proteins for further analysis or research.

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Lab products found in correlation

Bac to bac protocol, by Thermo Fisher Scientific (2 mentions) E coli strain dh10bac, by Thermo Fisher Scientific (2 mentions) Superdex g200 gel filtration column, by GE Healthcare (2 mentions) Pfastbachtb vector, by Thermo Fisher Scientific (2 mentions) Phosphodiesterase 1 from crotalus adamanteus venom, by Merck Group (1 mentions) Isopropyl β d 1 thiogalactopyranoside (iptg), by Merck Group (1 mentions) Superdex 200, by GE Healthcare (1 mentions) Monoclonal anti polyhistidine antibody, by Merck Group (1 mentions) Incomplete freund s adjuvant, by Merck Group (1 mentions) Complete freund s adjuvant, by Merck Group (1 mentions) Bca protein assay kit, by Bio-Rad (1 mentions)

7 protocols using nickel affinity chromatography column

Purification and Enzymatic Assays of DisA and YybT

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DisA and YybT were
expressed in BL21(DE3)
and purified by Nickel-affinity chromatography column (GE Healthcare).
DisA was dialyzed into a 10 mM Tris-HCl, pH 8.0, and 100 mM NaCl solution,
and YybT was dialyzed into a 50 mM Tris-HCl, pH 8.0, and 150 mM NaCl
solution. Phosphodiesterase I from Crotalus adamanteus venom (snake venom phosphodiesterase, SVPD) was purchased from Sigma-Aldrich.
For the c-di-AMP synthesis assay, DisA (10 μM) was added to
100 μM ATP in 40 mM Tris-HCl, pH 7.5, 100 mM NaCl, and 10 mM
MgCl₂ at 30 °C.^{7 (link)} For the
c-di-AMP cleavage assay, YybT (10 μM) in 100 mM Tris-HCl, pH
8.3, 20 mM KCl, 500 μM MnCl₂, and 1 mM DTT^{17 (link)} or SVPD (1 mg/mL) in 50 mM Tris-HCl, pH 8.8,
and 15 mM MgCl₂ was used to cleave c-di-AMP (100 μM)
at 37 °C. Reactions were stopped by heating up to 95 °C
for 5 min, and the precipitated proteins were removed by centrifugation.
KBr and coralyne were added to the sample to give final concentrations
of 250 mM and 10 μM for KBr and coralyne, respectively. The
sample was incubated at 4 °C for 12 h.

Zhou J., Sayre D.A., Zheng Y., Szmacinski H, & Sintim H.O. (2014). Unexpected Complex Formation between Coralyne and Cyclic Diadenosine Monophosphate Providing a Simple Fluorescent Turn-on Assay to Detect This Bacterial Second Messenger. Analytical Chemistry, 86(5), 2412-2420.

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EpCAM Extracellular Domain Expression

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The pQE30-EpCAM plasmid was used to express the extracellular domain of EpCAM in M15 E. coli. Cells were grown at 37°C in a shaking incubator (220 rpm) until the culture reached an OD₆₀₀ of 0.6–0.8. Protein expression was induced for 7 h at 30°C by adding IPTG (Sigma) at a final concentration of 500 nM. The harvested pellet was ultrasonicated and analyzed via SDS-PAGE Coomassie Blue staining and Western blot. The protein from the supernatant was purified with a Nickel-affinity chromatography column, in accordance with the manufacturer's instruction (GE).

Lv M., Qiu F., Li T., Sun Y., Zhang C., Zhu P., Qi X., Wan J., Yang K, & Zhang K. (2015). Construction, Expression, and Characterization of a Recombinant Immunotoxin Targeting EpCAM. Mediators of Inflammation, 2015, 460264.

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Purification of N-terminal Truncated SUVH9

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The N-terminal truncated SUVH9 (residues 134 - 650) was cloned into a pFastBacHT B vector (Invitrogen), which fuses a hexa-histidine tag followed by a TEV cleavage site to the N-terminus of the target gene. The plasmid was transformed into E. coli strain DH10Bac (Invitrogen) to generate the bacmid. Baculovirus was generated by transfecting Sf9 cells with the bacmid following standard Bac-to-Bac protocol (Invitrogen). The harvested virus was subsequently used to infect the suspended Hi5 cell for recombinant protein expression. The recombinant protein was first purified using nickel affinity chromatography column (GE Healthcare). The hexa-histidine tag was cleaved by TEV protease. The target protein was further purified using a Q sepharose column and a Superdex G200 gel filtration column (GE Healthcare). The purified protein was concentrated to 15 mg/ml and stored at −80°C.

Johnson L.M., Du J., Hale C.J., Bischof S., Feng S., Chodavarapu R.K., Zhong X., Marson G., Pellegrini M., Segal D.J., Patel D.J, & Jacobsen S.E. (2014). SRA/SET domain-containing proteins link RNA polymerase V occupancy to DNA methylation. Nature, 507(7490), 124-128.

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Purification of N-terminal Truncated SUVH9

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Purification of Human Mini-TyrRS Protein

Cited 1 time

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Human TyrRS was purified as previously described.^{7,36,37 (link)} The plasmid of mini-TyrRS (amino acids 1–341) as a gift from Paul Schimmel. Briefly, this plasmid was transformed into E. coli Arctic Express (DE3) cells. The cells were cultured at 37 °C in fresh LB liquid medium (5 g of NaCl, 10 g of bactotrypton, and 10 g of yeast extract per litre). Isopropyl-β-d-1-thiogalactopyranoside (IPTG, 0.5 mM) was used to induce expression when attenuance D_{600 nm} was 1, and the culture was grown for 20 h at 16 °C. Cells were collected by centrifugation at 4000 rpm min⁻¹ for 15 min at 4 °C and then re-suspended in lysis buffer (50 mM Hepes-Na, pH 7.5, 5 mM imidazole pH 7.5, 400 mM NaCl, 1× PMSF, and 2 mM BME). Cells were lysed using a cell disrupter. After centrifugation at 14 000 rpm min⁻¹ for 25 min at 4 °C, the supernatant was loaded onto a nickel-affinity chromatography column (GE-Healthcare) pre-equilibrated with lysis buffer. The protein was eluted with a linear gradient of imidazole (0–300 mM). The desired fractions were pooled, concentrated and subjected to gel-filtration (Superdex 200; GE-Healthcare) chromatography and the protein peak corresponding to homogenous protein in buffer (20 mM Hepes-Na, pH 7.5, 50 mM NaCl, and 2 mM BME) was collected (see Fig. S3†). The quality of the protein purification was validated by SDS-PAGE analysis (see Fig. S4†).

Huang S., Wang X., Lin G., Cheng J., Chen X., Sun W., Xiang R., Yu Y., Li L, & Yang S. (2019). Discovery of human TyrRS inhibitors by structure-based virtual screening, structural optimization, and bioassays. RSC Advances, 9(16), 9323-9330.

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SDF-1α Protein Expression and Purification

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The native human SDF-1α (NAT-SDF-1α) gene was amplified from human fibroblast cDNA by polymerase chain reaction. CBD-SDF-1α was constructed by incorporating the collagen-binding domain (CBD) into the C-terminus of NAT-SDF-1α. The gene sequence of SDF-1α was separated from the CBD sequence to preserve its activity by a linker (amino acid sequence: GSAGSAAGSGG). NAT-SDF-1α or CBD-SDF-1α was inserted into the pET28a vector (Novagen, Madison, WI), and then transformed into the BL21 strain (DE3) of Escherichia coli (Novagen, Madison, WI) for protein expression. Both proteins containing a 6 × histidine tag were purified by a Nickel affinity chromatography column (GE Healthcare) and identified by Tricine SDS-PAGE and western blotting using an anti-polyhistidine monoclonal antibody (1:3000; Sigma-Aldrich, St. Louis, MO, USA).

Sun J., Zhao Y., Li Q., Chen B., Hou X., Xiao Z, & Dai J. (2016). Controlled Release of Collagen-Binding SDF-1α Improves Cardiac Function after Myocardial Infarction by Recruiting Endogenous Stem Cells. Scientific Reports, 6, 26683.

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Polyclonal Antibody Production against Toxoplasma SIR2

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To obtain the polyclonal antibodies against recombinant T. gondii SIR2 protein (rTgSIR2), the E. coli BL21 (DE3) was induced to express rTgSIR2 by isopropyl β-d-thiogalactoside (IPTG, Sangon Biotech, Shanghai, China). Purified by nickel-affinity chromatography column (GE Healthcare, Marlborough, MA, USA), the eluted rTgSIR2 was analyzed by 12% SDS-PAGE electrophoresis and quantified by BCA protein assay kit (Bio-Rad, Hercules, CA, USA). The purified rTgSIR2 was stored at −80 °C until use. To generate polyclonal antibodies against rTgSIR2, SD rats were subcutaneously vaccinated with 200 μg of rTgSIR2 emulsified with an equal volume of complete Freund’s adjuvant (Sigma, Saint Louis, MO, USA). Fourteen days later, booster vaccinations were conducted with 200 μg of rTgSIR2 emulsified with an equal volume of incomplete Freund’s adjuvant (Sigma, Saint Louis, MO, USA) through the subcutaneous injection. The booster vaccinations were performed five times in total and the time interval between two vaccinations was seven days. Seven days after the last vaccination, the blood was collected at the eye socket and the serum was harvested.

Yu Z., Lu Y., Cao W., Aleem M.T., Liu J., Luo J., Yan R., Xu L., Song X, & Li X. (2021). Nano DNA Vaccine Encoding Toxoplasma gondii Histone Deacetylase SIR2 Enhanced Protective Immunity in Mice. Pharmaceutics, 13(10), 1582.

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