Ni nta affinity chromatography

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Ni-NTA affinity chromatography is a technique used for the purification of proteins that have a histidine-tag (His-tag) attached to them. The Ni-NTA (Nickel-Nitrilotriacetic Acid) resin binds to the His-tag, allowing the target protein to be separated from other components in the sample. This method is widely used in the purification of recombinant proteins expressed in various host systems.

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Lab products found in correlation

Ni nta agarose, by GE Healthcare (1 mentions) Hitrap heparin high performance, by GE Healthcare (1 mentions) Monos chromatography, by GE Healthcare (1 mentions) Superdex 200 column, by GE Healthcare (1 mentions) E coli rosetta de3 competent cells, by Thermo Fisher Scientific (1 mentions) Pet28b vector, by Thermo Fisher Scientific (1 mentions) Prescission protease, by GE Healthcare (1 mentions) Nanodrop 2000c spectrophotometer, by Thermo Fisher Scientific (1 mentions) Sonifier 450, by Emerson (1 mentions) Pegfp n1 plasmid, by BD (1 mentions) Amylose affinity chromatography, by New England Biolabs (1 mentions) Pmal c, by New England Biolabs (1 mentions) Complete edta free protease inhibitor tablet, by Roche (1 mentions) Escherichia coli bl21 de3 cells, by New England Biolabs (1 mentions) Adp glo kinase assay kit, by Promega (1 mentions) Envision multimode plate reader, by PerkinElmer (1 mentions) Pherastar fsx microplate reader, by BMG Labtech (1 mentions) Minimum essential medium (mem), by Thermo Fisher Scientific (1 mentions) Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions) Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)

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Ni-NTA affinity chromatography column HiTrap columns for Ni-NTA affinity chromatography Ni-NTA affinity Ni-NTA chromatography Ni affinity chromatography Ni-NTA

20 protocols using ni nta affinity chromatography

Purification of Recombinant YB-1 and PARP1 Proteins

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Recombinant YB-1 and its mutants [YB-1 (Δ1), YB-1 (Δ1-2), or YB-1 (Δ1-2-3)] were overexpressed in Escherichia coli BL21 (DE3) and purified. YB-1 was purified by Ni-NTA affinity chromatography (GE Healthcare United States, catalog # GE17-5255-01), Mono-S chromatography (GE Healthcare, United States catalog # GE17-5168-01), and Superdex 16/600 chromatography (GE Healthcare, United States, catalog # GE28-9893-35) as described earlier (Alemasova et al., 2017 (link)). YB-1 mutants were purified by Ni-NTA and Mono-S chromatography.
Recombinant wild-type (wt) PARP1 and mutants PARP1^Y986S, PARP1^Y986H, and PARP1^G972R were overexpressed in E. coli Rosetta (DE3)pLysS (Novogen, catalog # 70956-3) and purified by Ni-NTA agarose (GE Healthcare United States, catalog # GE17-5255-01) affinity chromatography, HiTrap Heparin High Performance (GE Healthcare, United States, catalog # GE17-0407-01) affinity chromatography, and deoxyribonucleic acid−cellulose (single-stranded calf thymus DNA) (Sigma-Aldrich, United States, catalog #D8273) affinity chromatography as described previously (Sukhanova et al., 2004 (link)).
Yeast nicotinamide mononucleotide adenylyltransferase (NMNAT) was kindly provided by Dr. S.I. Shram (Institute of Molecular Genetic Russian Academy of Science, Moscow, Russia).

Naumenko K.N., Sukhanova M.V., Hamon L., Kurgina T.A., Anarbaev R.O., Mangerich A., Pastré D, & Lavrik O.I. (2022). The C-Terminal Domain of Y-Box Binding Protein 1 Exhibits Structure-Specific Binding to Poly(ADP-Ribose), Which Regulates PARP1 Activity. Frontiers in Cell and Developmental Biology, 10, 831741.

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Purification and Characterization of APC Mutants

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The DNA encoding APC (303–739), APC (407–751), and site-directed mutants (W593A, N641A) were cloned into pET28a expression vector and expressed as His-tagged fusion proteins in Escherichia coli (E. coli) strain BL21 (DE3)^{16 (link)}. The primers were provided in Supplementary Table 8. The cells were harvested and resuspended in lysis buffer (25 mM Tris-HCl, pH 8.0, 300 mM NaCl, and 20 mM imidazole). After sonication, the cell lysates were centrifuged and purified by Ni-NTA affinity chromatography (GE Healthcare). The purity of APC (residues 303–739) was confirmed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) analysis.

Zhong J., Guo Y., Lu S., Song K., Wang Y., Feng L., Zheng Z., Zhang Q., Wei J., Sang P., Shi Y., Cai J., Chen G., Liu C.Y., Yang X, & Zhang J. (2022). Rational design of a sensitivity-enhanced tracer for discovering efficient APC–Asef inhibitors. Nature Communications, 13, 4961.

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Overproduction and Purification of CpsR Protein

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The CpsR protein was overproduced in E. coli BL21 (DE3) using the expression plasmid pW28-cpsR and purified by nickel chelate affinity chromatography according to the established protocol46 (link). Expression was induced at an OD₆₀₀ of 2.0 by addition of 1 mM isopropyl-D-thiogalactoside (IPTG) at 20 °C. Eight hours after induction, cells were harvested by centrifugation, washed once with the appropriate disruption buffer, and disrupted by ultrasonication. The recombinant protein was purified by nickel-nitrilotriacetic acid (Ni-NTA) affinity chromatography (GE Healthcare). Protein concentrations were determined as described above, and the purified proteins were stored at −80 °C until use.

Wu K., Xu H., Zheng Y., Wang L., Zhang X, & Yin Y. (2016). CpsR, a GntR family regulator, transcriptionally regulates capsular polysaccharide biosynthesis and governs bacterial virulence in Streptococcus pneumoniae. Scientific Reports, 6, 29255.

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Escherichia coli Protein Expression and Purification

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All proteins were expressed in Escherichia coli Bl21-Gold (DE3) cells (Novagen). For native proteins, cells were cultured in LB medium at 310 K to an OD600 of 0.8–1.0, subsequently shifted to 289 K and induced with 0.4 mM isopropyl β-D-thiogalactopyranoside (IPTG) for 24 h. For Se-Met labeled proteins, cells were cultured in LeMaster and Richards minimal medium (LR medium) at 310 K to an OD600 of 0.8–1.0, then transferred to LR medium that contained Val, Ile, Leu, Phe, Trp, Thr, Lys at the concentration of 50 mg/l and Se-Met at the concentration of 60 mg/l. After 30 min at 310 K, cells were shifted to 289 K and induced with 0.4 mM IPTG for 24 h. All proteins were purified by Ni-NTA affinity chromatography (GE Healthcare) after lysing cells by sonication in 20 mM Tris (pH 8.0) and 500 mM NaCl. After clarification at 13 000 rpm in an R22A2 Hi-tachi rotor (Hitachi, himac CR22G, Japan) for 30 min at 277 K, the supernatants were eluted in buffer containing 30–500 mM imidazole (pH 7.5) at 293 K. The eluted His-tag proteins were further purified using a Superdex 200 column (GE Healthcare) in buffer containing 20 mM Tris (pH 7.5) and 200 mM NaCl with no reducing agent at 293 K.

Bao H., Wang N., Wang C., Jiang Y., Liu J., Xu L., Wu J, & Shi Y. (2017). Structural basis for the specific recognition of 18S rRNA by APUM23. Nucleic Acids Research, 45(20), 12005-12014.

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Recombinant Schistosome Acetylcholinesterase Expression

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Recombinant SjAChE was expressed in the Escherichia coli expression system. A fragment encoding the SjAChE (G71-Y537) was amplified by PCR and ligated into the pET28b vector (Invitrogen) by using forward (5′-CGGGATCCGGGAATTTACTGTGGACAACGTG-3′ with BamHI restriction site underlined) and reverse (5′-CGCTCGAGATAACCATGCATTGTACCAGTCC-3′ with Xhol restriction site underlined) primers. E. coli Rosetta™ (DE3) competent cells (Invitrogen) were transformed with the recombinant plasmids for expression and purification as described [30 (link)]. The expressed SjAChE protein was purified from the E. coli lysate using Ni-NTA affinity chromatography (GE Healthcare Life Science, Chicago, IL, USA) under denaturing conditions, using 6 M guanidine-HCl and then refolding in buffer (300 mM NaCl, 50 mM NaH₂PO₄, 8% w/v sucrose, PH 4.5). Residual endotoxin was removed from purified proteins as described [30 (link)] and assessed by using an Endotoxin (E. coli) Standards kit (Lonza, Anaheim, CA, USA). The purified rSjAChE was used in mice vaccine/challenge experiments.

You H., Liu C., Du X., Nawaratna S., Rivera V., Harvie M., Jones M, & McManus D.P. (2018). Suppression of Schistosoma japonicum Acetylcholinesterase Affects Parasite Growth and Development. International Journal of Molecular Sciences, 19(8), 2426.

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Overexpression and Purification of Tfu_0875

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The E. coli BL21(DE3) harboring Tfu_0875 and its mutants were grown on LB medium containing 50 μg mL⁻¹ kanamycin at 37 °C and 250 rpm overnight as seeds. These seeds were exponentially growing to OD₆₀₀ = 1.0 at 37 °C, and then were treated with 0.8 mM isopropyl β‐d‐1‐thiogalactopyranoside (IPTG) to induce. These cells were cultured for 12–14 h at 25 °C. All cells were collected by centrifugation at 4500g for 5 min and washed with buffer A (50 mM Tris-HCl, pH 7.4, and 200 mM NaCl) twice, and then disrupted by ultrasonication on ice. Then the lysate was centrifuged at 6000 g for 30 min, and the supernatant was filtered by a 0.45 μm membrane filtration. The collected supernatant was loaded onto Ni-NTA affinity chromatography (5 mL, GE health, ShangHai, China). The target protein was eluted with buffer A (50 mM Tris-HCl, pH 7.4, and 200 mM NaCl) with 20 mM imidazole and buffer B (50 mM Tris-HCl, pH 7.4, 200 mM NaCl, and 200 mM imidazole) [9 (link)]. All plasmid schematic diagrams were illustrated in Fig. S1. The protein concentration was determined by Bradford's method (Sangon Biotech, Shanghai, China).

Liu L., Liu S., Hu X., Zhou S, & Deng Y. (2024). Enhancing the activity and succinyl-CoA specificity of 3-ketoacyl-CoA thiolase Tfu_0875 through rational binding pocket engineering. Synthetic and Systems Biotechnology, 9(3), 558-568.

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Recombinant Protein Expression and Purification

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The recombinant plasmid pET28a-N was transformed into Escherichia coli strain BL21 (TransGen Biotech, Beijing, China) for protein expression. The recombinant pET28a-N protein was produced by inducing the positive bacterial strain with 0.1% isopropyl β-D-1-thiogalactopyranoside (IPTG) for 6 hours at 37°C. The harvested cells were lysed before the recombinant protein was purified using Ni-NTA affinity chromatography (GE Healthcare, Chicago, IL, USA) following the manufacturer’s instructions (17 (link)). The purified recombinant protein was analyzed with an anti-His-tagged mAb, as well as positive PRRSV serum, by Western blotting (18 (link)).

Cheng Y., Wu M., Xiao L., Zhang M., Huang B., Cong F, & Yi L. (2023). Identificationof a novel linear epitope on the porcine reproductive and respiratory syndrome virus nucleocapsid protein, as recognized by a specific monoclonal antibody. Frontiers in Immunology, 14, 1165396.

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Cloning and Purification of Adk from Bombyx mori

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The DNA fragment encoding Adk was obtained from the cDNA library by PCR from the midgut of Bombryx mori strain Dazao using primer sets (5′-ATGGCACCGGCCGCTGC-3′ and 5′-TTACAAA GCAGACCGTGCTCTGCTG-3′). The amplification product was gel-purified, recovered, and inserted into plasmid vectors pSKB2. The bacterial transformants containing error-free inserts were identified. Adk was expressed in Escherichia coli BL21(DE3) and purified by Ni-NTA affinity chromatography (GE Healthcare, Chicago, IL, USA). The fused polyhistidine tag was cleaved by Prescission protease (GE Healthcare, USA) and removed as described by Liu et al. [16 (link)]. Protein concentration was determined using the extinction coefficient of 12,950 M⁻¹·L·cm⁻¹ at 280 nm on a NanoDrop 2000C spectrophotometer (Thermo Fisher, Waltham, MA, USA).

Song K., Wang Y., Li Y., Ding C., Cai R., Tao G., Zhao P., Xia Q, & He H. (2019). A Convenient, Rapid, Sensitive, and Reliable Spectrophotometric Assay for Adenylate Kinase Activity. Molecules, 24(4), 663.

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Recombinant p53 Domain Purification

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The cDNA encoding different domains of p53 was subcloned into the pET28a vector, which was then transformed into E. coli BL21 (DE3) and the recombinant proteins were purified using Ni-NTA affinity chromatography (GE Healthcare).

Li C., Li W., Cheng X., Zhang D., Sun X., Zhou J., Zhou Y., Huang Y., Xia X., Ma Q, & Su Z. (2020). P55PIK Regulates P53-Dependent Apoptosis in Cancer Cells by Interacting with P53 DNA-Specific Domain. OncoTargets and therapy, 13, 5177-5190.

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Soluble Expression and Purification of EGFP

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The gene encoding enhanced GFP was cloned from pEGFP-N1 plasmid (BD Clontech, Palo Alto, CA) and inserted into pET28a expression vector and transferred into BL21 (DE3). When the cells were grown in LB medium at 37°C with the absorbance at 600 nm (OD 600) reached 0.8, a final concentration of 1 mM IPTG was added to induce the EGFP soluble expression at 16°C for another 16 hours, otherwise insoluble form was induced at 37°C for 4 hours. For EGFP soluble expression, the cells were harvested at 5,000 g at 4°C for 20 minutes and resuspended in 30 ml of PBS buffer (137 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄ · 12H₂O, 2 mM KH₂PO₄, pH 8.0), and then lysed by sonication on ice (Branson Sonifier 450, USA). The lysate was centrifuged at 12,000 g at 4°C for 20 minutes. The clarified supernatant was collected and purified by Ni-NTA affinity chromatography (GE healthcare) following the instructions. For insoluble expression, cells were harvested at 5,000 g at 4°C for 20 minutes and the pellets were stored at −20°C.

Qi X., Sun Y, & Xiong S. (2015). A single freeze-thawing cycle for highly efficient solubilization of inclusion body proteins and its refolding into bioactive form. Microbial Cell Factories, 14, 24.

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