Ni nta agarose column

Manufactured by GE Healthcare

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Ni-NTA agarose column is a chromatography resin used for the purification of histidine-tagged recombinant proteins. The resin consists of nickel-nitrilotriacetic acid (Ni-NTA) immobilized on agarose beads, which bind to the histidine tag on the target protein. This allows for selective capture and purification of the desired protein from complex mixtures.

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Lab products found in correlation

Hitrap, by Cytiva (3 mentions) High performance liquid chromatography (hplc), by Shimadzu (1 mentions) Bca protein quantification kit, by CWBIO (1 mentions) Hitrap q column, by GE Healthcare (1 mentions) Fast mutagenesis system, by Transgene (1 mentions) Protein g sepharose 4b column, by GE Healthcare (1 mentions) Pqe30 expression vector, by Qiagen (1 mentions) Geneart, by Thermo Fisher Scientific (1 mentions) Prset a expression vector, by Thermo Fisher Scientific (1 mentions) Superdex 200 10 300 size exclusion column, by GE Healthcare (1 mentions) Bl21 de3 cells, by New England Biolabs (1 mentions) Irdye 800cw goat anti rabbit secondary antibody, by LI COR (1 mentions) Irdye 680rd goat anti mouse secondary antibody, by LI COR (1 mentions) Clonexpress 2 one step cloning kit, by Vazyme (1 mentions) Hitrap heparin column, by GE Healthcare (1 mentions) Superdex 200 column, by GE Healthcare (1 mentions) Atpase gtpase activity assay kit, by Merck Group (1 mentions) Mono q 5 50 column, by GE Healthcare (1 mentions) Superdex 16 600 column, by GE Healthcare (1 mentions)

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Ni-NTA column (150 citations) Ni-NTA agarose (51 citations) Ni-NTA (45 citations) Agarose (6 citations) Ni-agarose column (2 citations)

12 protocols using ni nta agarose column

Purification of Recombinant Proteins

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All chemicals and reagents were of the highest purity commercially available from Sigma-Aldrich (St. Louis, MO, USA) and Merck (Darmstadt, Germany). Ni-NTA-agarose columns were obtained from GE Healthcare (Little Chalfont, UK).

Lienhart W.D., Strandback E., Gudipati V., Koch K., Binter A., Uhl M.K., Rantasa D.M., Bourgeois B., Madl T., Zangger K., Gruber K, & Macheroux P. (2017). Catalytic competence, structure and stability of the cancer-associated R139W variant of the human NAD(P)H: quinone oxidoreductase 1 (NQO1). The FEBS journal, 284(8), 1233-1245.

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Purification of Recombinant Proteins

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Purification of a Recombinant Protein

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Following induction using methanol for 96 h, the supernatant of the high-expressing clone was collected and dialyzed against a binding buffer (20 mM phosphate buffer, 300 mM NaCl, pH 7.4) overnight using a 10 kDa cutoff dialysis membrane (Millipore, Billerica, MA, USA). Subsequent to filtering through 0.22 um filters (Millipore, Billerica, MA, USA), the supernatants were added to a nickel-nitrilotriacetic acid (Ni-NTA) agarose column (GE, Boston, FA, USA) with 1 mL/min flow velocity, and then the column was washed with ten column volumes of binding buffer. The interest protein bound to the column was eluted with 250 mM imidazole in a binding buffer. The elution fraction containing the interest protein was dialyzed against PBS overnight to remove the imidazole. Finally, the protein was concentrated using ultrafiltration and the concentration of the purified protein was measured using a BCA protein quantification kit (CW biotech, Beijing, China) and HPLC (Shimadzu, Tyoto, Japan) was used to determine the purity of this BsDb, as described in our previous study [47 (link)].

Xiong C., Mao Y., Wu T., Kang N., Zhao M., Di R., Li X., Ji X, & Liu Y. (2018). Optimized Expression and Characterization of a Novel Fully Human Bispecific Single-Chain Diabody Targeting Vascular Endothelial Growth Factor165 and Programmed Death-1 in Pichia pastoris and Evaluation of Antitumor Activity In Vivo. International Journal of Molecular Sciences, 19(10), 2900.

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Construction and Purification of Mutant Protein Clones

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The 19 mutant clones were constructed by a PCR-based strategy using the Fast Mutagenesis System (TransGen Biotech, Beijing, China). The corresponding primers were listed in Supplementary Table S1. Each mutant clone, verified by DNA sequencing, was transferred into BL21(DE3) and expressed using the same procedure for the wild type clone. The mutant protein was purified tandemly by using the Ni-NTA agarose column (GE Healthcare), the HiTrap Q column (GE Healthcare) and the Superdex 200 Increase prepacked column (GE Healthcare).

Liu F., Su Z., Chen P., Tian X., Wu L., Tang D.J., Li P., Deng H., Ding P., Fu Q., Tang J.L, & Ming Z. (2021). Structural basis for zinc-induced activation of a zinc uptake transcriptional regulator. Nucleic Acids Research, 49(11), 6511-6528.

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Generation of Anti-Human HAT-L4 Antibodies

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A cDNA fragment encoding human HAT-L4 protease domain (amino acids 196–433 based on NCBI accession number NP_997290.2) was amplified from the human leukemia cell line MEG-01 by RT-PCR. The PCR fragment was sequenced and inserted into pQE30 expression vector encoding a C-terminal His tag (Qiagen). Recombinant HAT-L4 protein was expressed in Escherichia coli, purified with a Ni-NTA agarose column (GE Healthcare Life Sciences), and verified by SDS-PAGE and Coomassie blue staining. The purified HAT-L4 protein was used to immunize BALB/c mice to make monoclonal antibodies. Positive hybridoma cells were identified by ELISA using the purified recombinant HAT-L4 protein and used to produce ascites in mice. Monoclonal antibodies were purified from the ascites using a protein-G-Sepharose 4B column (GE Healthcare Life Sciences) and analyzed by Western blotting, immunostaining and flow cytometry. In Western blotting and immunohistochemical analyses, the antibodies recognized human HAT-L4 protein but not mouse HAT-L4 protein.

Zhang Z., Hu Y., Yan R., Dong L., Jiang Y., Zhou Z., Liu M., Zhou T., Dong N, & Wu Q. (2017). The Transmembrane Serine Protease HAT-like 4 Is Important for Epidermal Barrier Function to Prevent Body Fluid Loss. Scientific Reports, 7, 45262.

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Purification of Recombinant Tg14-3-3 Protein

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The Tg14-3-3 coding sequences were synthesized (GeneArt, LifeTechnologies) and expressed through a pRSET-A expression vector (LifeTechnologies) with a N-Terminal His₆-tag in T7express competent cells (NEB) at 37°C for 4h in the presence of 0.4 mM IPTG. The protein was purified as previously described (Nurmi et al., 2006 (link)). Briefly rTg14-3-3 was isolated through a NiNTA agarose column (GE Healthcare) from the clarified lysate (PBS, 0.05% Triton-X100, 10 mM Imidazol, 1 mM PMSF, 2 mM MgCl₂, 50 μg/ml DNAse I). The eluted protein was further purified using HiTrap MonoQ chromatography (GE Healthcare) in 20 mM Tris pH 7.0, 50 mM NaCl, 2 mM DTT, with linear NaCl gradient elution. Final purification was performed on a Superdex 200 10/300 size-exclusion column (GE Healthcare) in PBS, 2 mM DTT and 2 mM EDTA. The His-tag was removed by digestion with 0.2 U of Enterokinase (LifeTechnologies) per 40 μg 14-3-3 and incubating according to the manufacturer’s instruction followed by a pass over NiNTA agarose and gel filtration chromatography.

Weidner J.M., Kanatani S., Uchtenhagen H., Varas-Godoy M., Schulte T., Engelberg K., Gubbels M.J., Sun H.S., Harrison R.E., Achour A, & Barragan A. (2016). Migratory activation of parasitized dendritic cells by the protozoan Toxoplasma gondii 14-3-3 protein. Cellular microbiology, 18(11), 1537-1550.

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Expression and Purification of His6-Tagged TglA

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For peptide expression, E. coli BL21(DE3) cells (New England Biolabs) expressing N-terminal His₆-tagged TglA from His₆-TglA-pRSF-Duet-1 were grown with 50 μg/mL kanamycin in autoinduction (AI) media (for 1 L total: 10 g bacto tryptone, 5 g yeast extract, 5 g NaCl, 3 g KH₂PO₄, 6 g Na₂HPO₄) with 1X AI sugar solution containing 0.5% (vol/vol) glycerol, 0.05% (w/vol) glucose, and 0.2% (w/vol) lactose (20 mL of 50× stock)). Cultures were shaken at 37 °C for 6-7 h following inoculation with 1 mL of saturated culture. Cells were harvested and lysed by sonication. Peptides were purified by immobilized metal affinity chromatography (IMAC). The lysate was clarified by centrifugation at 29,000 rcf and applied to a 5 mL NiNTA-agarose column (GE Healthcare) using a peristaltic pump. The immobilized peptide was washed with 5 column volumes (CV) of 90% lysis buffer (50 mM HEPES, 100 mM NaCl, pH 7.5), 10% elution buffer (50 mM HEPES, 100 mM NaCl, 500 mM imidazole, pH 7.5; final imidazole concentration in wash: 50 mM) and eluted with 100% elution buffer. The elution fraction was concentrated using a 3 kDa MWCO Amicon spin filter and washed with 10-20 CV of deionized water to remove imidazole. Crude peptide was desalted with a VYDAC^® Bioselect C4 cartridge. Peptide elution fractions were lyophilized.

Zhang Z, & van der Donk W.A. (2019). Non-ribosomal Peptide Extension by a Peptide Amino-acyl tRNA Ligase. Journal of the American Chemical Society, 141(50), 19625-19633.

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Protein Expression and Purification

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All chemicals used in this study were purchased from Sigma-Aldrich unless stated otherwise. Antibodies were obtained from Abcam (rabbit anti-lipoic acid antibody, goat anti-mouse secondary antibody, HRP conjugated) and LI-COR (IRDye 680RD goat anti-mouse secondary antibody, IRDye 800CW goat anti-rabbit secondary antibody). PCR amplification was performed using Taq (New England Biolabs) PrimeSTAR HS (Premix) (TaKaRa) polymerase. Restriction enzymes and T4 DNA ligase were supplied by TaKaRa. A ClonExpress II One Step Cloning Kit was purchased from Vazyme. GE Healthcare provided a Ni-NTA agarose column.

Jin J., Chen H., Wang N., Zhu K., Liu H., Shi D., Xin J, & Liu H. (2021). A Novel Lipoate-Protein Ligase, Mhp-LplJ, Is Required for Lipoic Acid Metabolism in Mycoplasma hyopneumoniae. Frontiers in Microbiology, 11, 631433.

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Expression and Purification of SARS-CoV-2 M^pro

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The plasmid pGEX-6p-1-MPro encoding SARS-CoV-2 M^pro (ORF1ab polyprotein residues 3264–3569, GenBank code: MN908947.3) with Escherichia coli (E. coli) codon usage was kindly provided by Prof. Rolf Hilgenfeld (Institute of Biochemistry, Center for Structural and Cell Biology in Medicine, University of Lübeck, Germany). The protocol for expression and purification of the M^pro was performed as reported by Zhang et al. (2020) (link), with some modifications. The bacterial pellets were resuspended in 15 mL of cold Resuspension buffer [20 mM Tris HCl pH 7.5, 0.5 M NaCl, 10% glycerol, 1 mg/mL lysozyme, 1.15 mg/mL complete EDTA-free protease inhibitor cocktail (Roche)]. Cells were lysed by two freeze/thaw cycles and then sonicated. The lysate was clarified by centrifugation at 13,000 rpm at 4 °C for 45 min. The supernatant was then loaded onto an NiNTA Agarose column (GE Healthcare) equilibrated with 10 mL of Binding buffer (20 mM Tris HCl pH 7.5, 0.5 M NaCl, 10% glycerol). The column was washed with 20 mL Wash buffer (20 mM Tris HCl pH 7.5, 0.5 M NaCl, 10% glycerol, 25 mM imidazole) and elution was performed using 6 mL of elution buffer (20 mM Tris HCl pH 7.5, 0.5 M NaCl, 10% glycerol, 125 mM imidazole). Fractions were dialyzed against 20 mM Tris-HCl pH 8.0, 150 mM NaCl, 30% (vol/vol) glycerol, 5 mM DTT and stored at −80 °C.

Mercorelli B., Desantis J., Celegato M., Bazzacco A., Siragusa L., Benedetti P., Eleuteri M., Croci F., Cruciani G., Goracci L, & Loregian A. (2022). Discovery of novel SARS-CoV-2 inhibitors targeting the main protease Mpro by virtual screenings and hit optimization. Antiviral Research, 204, 105350.

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Affinity Purification of Recombinant Protein

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The cells were harvested by centrifugation at 4200 rpm for 15 min, resuspended with 30 ml lysis buffer containing 20 mM Tris–HCl (pH 8.0), 150 mM NaCl, 30 mM imidazole, and then homogenized using a low-temperature ultra-high-pressure cell disrupter (JNBIO, Guangzhou, China). After centrifugation at 12 000 rpm for 1 h, the resulting supernatant was loaded onto a Ni-NTA agarose column (GE Healthcare). The eluted protein was subjected onto a HiTrap Heparin column (GE Healthcare) equilibrated with 20 mM Tris–HCl (pH 8.0), 75 mM NaCl. The protein was eluted by a linear NaCl gradient and further purified by size exclusion chromatography using Superdex 200 columns (GE Healthcare) in a buffer containing 20 mM Tris (pH 8.0) and 150 mM NaCl. The protein was concentrated to approximate 10 mg/ml and stored at −80°C.

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