Nickel nitrilotriacetic acid ni nta agarose

Manufactured by Qiagen

Sourced in United States, Germany

Nickel-nitrilotriacetic acid (Ni-NTA) agarose is a chromatographic resin used for the purification of recombinant proteins with a poly-histidine tag. It utilizes the high affinity interaction between nickel ions and histidine residues to selectively capture and isolate the target protein from complex mixtures.

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Lab products found in correlation

Coomassie plus protein assay reagent, by Thermo Fisher Scientific (1 mentions) 13c2 oxalic acid, by Cambridge Isotopes (1 mentions) Polypropylene column, by Qiagen (1 mentions) Ultra centrifugal filters, by Merck Group (1 mentions) Vent dna polymerase and restriction enzymes, by New England Biolabs (1 mentions) Oligonucleotide, by Integrated DNA Technologies (1 mentions) Zymolase 20t, by MP Biomedicals (1 mentions) Diamide, by Merck Group (1 mentions) Horseradish peroxidase, by Merck Group (1 mentions) Isopropyl β d thiogalactopyranoside, by Fujifilm (1 mentions) Superscript 3 reverse transcriptase, by Thermo Fisher Scientific (1 mentions) Micro bca protein assay kit, by Thermo Fisher Scientific (1 mentions) Isogen ls, by Nippon Gene (1 mentions) Su 8 3050, by Nippon Kayaku (1 mentions) Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions) Disposable pd 10 desalting column, by GE Healthcare (1 mentions) P azido l phenylalanine azf, by Chem-Impex (1 mentions) Hitrap q hp anion exchange column, by GE Healthcare (1 mentions) Camp parameter assay kit, by R&D Systems (1 mentions) X tremegene hp dna transfection reagent, by Roche (1 mentions)

Spelling variants of this product

Ni-NTA agarose (1236 citations) Ni-NTA (356 citations) Nickel-nitrilotriacetic acid agarose (54 citations) Nickel-NTA agarose (33 citations) Ni-nitrilotriacetic acid (NTA) agarose (22 citations) Ni–nitrilotriacetic acid agarose (16 citations) Nickel agarose (14 citations) Nickel-NTA (13 citations) Nickel-nitrilotriacetic acid (11 citations) Nickel-nitrilotriacetic acid (Ni-NTA) (3 citations)

16 protocols using nickel nitrilotriacetic acid ni nta agarose

Isotope Analysis of Oxalate Compounds

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All chemicals and reagents
were purchased
from Fisher Scientific (Pittsburgh, PA) or Sigma-Aldrich (St. Louis,
MO), unless otherwise stated. [¹³C₂]Oxalic acid
(99% ¹³C) was purchased from Cambridge Isotope Laboratories
(Tewksbury, MA), and an oxalate assay kit was obtained from Trinity
Biotech USA (Jamestown, NY). Nickel-nitrilotriacetic acid agarose
(Ni-NTA) was supplied by Qiagen (Germantown, MD). DNA primers were
synthesized by Integrated DNA Technologies, Inc. (Coralville, IA),
and DNA sequencing was performed in the DNA Sequence Core at the University
of Michigan (Ann Arbor, MI). Protein concentrations were determined
using the CoomassiePlus Protein Assay reagent obtained from ThermoFisher
Scientific (Waltham, MA), and BT Chelex 100 resin was purchased from
Bio-Rad (Hercules, CA). ICP-MS measurements of metal content were
taken at the Center for Applied Isotope Studies at the University
of Georgia (Athens, GA).

Zhu W., Easthon L.M., Reinhardt L.A., Tu C., Cohen S.E., Silverman D.N., Allen K.N, & Richards N.G. (2016). Substrate Binding Mode and Molecular Basis of a Specificity Switch in Oxalate Decarboxylase. Biochemistry, 55(14), 2163-2173.

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Recombinant Protein Production Protocol

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All the chemicals were purchased from commercial sources and were either analytical grade or molecular grade. The growth media components were obtained from BD Difco (Franklin Lakes, NJ, USA) and Himedia (Mumbai, India). The amino acids, glutathione (GSH) (reduced), β‐nicotinamide adenine dinucleotide phosphate sodium salt hydrate (NADP), glucose 6‐phosphate (G6P), mevalonolactone and 6‐phosphogluconic dehydrogenase from yeast were obtained from Merck (Darmstadt, Germany). Zymolase‐20T was obtained from MP biomedicals (USA). Ultra centrifugal filters were obtained from Merck (Burlington, MA, USA). Oligonucleotides were obtained from Integrated DNA Technologies (IDT) and Merck (Bangalore, India). Vent DNA polymerase and restriction enzymes were obtained from New England Biolabs (Ipswich, MA, USA). Plasmid miniprep and gel/PCR clean‐up kits were purchased from Thermo Fisher Scientific (Waltham, MA, USA). The NADPH kit was obtained from Promega (Madison, WI, USA), nickel‐nitrilotriacetic acid agarose (Ni‐NTA), and polypropylene columns were obtained from Qiagen (Hilden, Germany).

Adusumilli S.H., Alikkam Veetil A., Choudhury C., Chattopadhyaya B., Behera D, & Bachhawat A.K. (2024). Glucose 6‐phosphate dehydrogenase variants increase NADPH pools for yeast isoprenoid production. FEBS Open Bio, 14(3), 410-425.

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Oxidative Stress Reagent Characterization

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H₂O₂, tBOOH, peroxynitrite, cumene hydroperoxide and 12(S)-hydroperoxy-5Z,8Z,10E,14Z eicosatetraenoic acid (12(S)HpETE) were purchased from Mallinckrodt Chemicals or Sigma. The concentration of H₂O₂ stock solutions was determined spectrophotometrically at 240 nm (ε_240 nm = 43.6 M⁻¹ cm⁻¹). The peroxynitrite concentration was determined at alkaline pH at 302 nm (ε_302 nm = 1.67 mM⁻¹ cm⁻¹) [14] (link). Concentrations of cumene hydroperoxide and 12(S)HpETE were calculated considering the manufactures specifications. Diamide, 1,4-dithiothreitol (DTT), diethylenetriaminepentaacetic acid (DTPA) and horseradish peroxidase were purchased from Sigma. Nickel-nitrilotriacetic acid agarose (Ni-NTA) was from Qiagen. HiTrap desalting columns were from Amersham Bioscience. All of the amino acids, glucose and yeast nitrogen base required for Hartwell's Complete (HC) yeast growth medium were purchased from Sigma. Flat-bottom 96 well microplates (product #353219) were from BD Biosciences.

Staudacher V., Trujillo M., Diederichs T., Dick T.P., Radi R., Morgan B, & Deponte M. (2017). Redox-sensitive GFP fusions for monitoring the catalytic mechanism and inactivation of peroxiredoxins in living cells. Redox Biology, 14, 549-556.

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Fluorescence-based Protein Quantification

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Phosphate-buffered saline (PBS; pH 7.4) solution and Micro BCA Protein Assay Kit were purchased from Thermo Fisher Scientific, Inc. (Waltham, MA). The Sylgard 184 silicone elastomer kit for polydimethylsiloxane (PDMS) microdevice fabrication was purchased from Dow Corning Toray Co., Ltd. (Tokyo, Japan). The PDMS included black silicon rubber to decrease the background signal of fluorescence. A negative photoresist (SU-8 3050) for PDMS microdevice fabrication was purchased from Nippon Kayaku Co., Ltd. (Tokyo, Japan). Isogen-LS was purchased from Nippon Gene (Tokyo, Japan). SuperScript III Reverse Transcriptase was purchased from Invitrogen (Carlsbad, CA). TaKaRa Ex Taq was purchased from TaKaRa (Shiga, Japan). Expression vector pET-32b and Rosetta-gamiTM 2 (DE3) pLysS were purchased from Novagen (San Diego, CA). Isopropyl-β-d-thiogalactopyranoside was purchased from Wako Pure Chemical Industries (Osaka, Japan). Nickel–nitrilotriacetic acid (Ni–NTA) agarose was purchased from Qiagen (Hilden, Germany). Lightning-Link ® Rapid Fluorescein was purchased from Innova Biosciences Co., Ltd. (Cambridge, United Kingdom).

Nishiyama K., Takeda Y., Maeki M., Ishida A., Tani H., Shigemura K., Hibara A., Yonezawa Y., Imai K., Ogawa H, & Tokeshi M. (2020). Rapid detection of anti-H5 avian influenza virus antibody by fluorescence polarization immunoassay using a portable fluorescence polarization analyzer. Sensors and Actuators. B, Chemical, 316, 128160.

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Chemoenzymatic Labeling of Proteins

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We purchased p-azido-l-phenylalanine (AzF; purity > 99%) from Chem-Impex International (Wood Dale, IL, USA). Nickel–nitrilotriacetic acid (Ni-NTA) agarose and propylene columns were obtained from Qiagen (Valencia, CA, USA). We purchased dibenzocyclooctyne-PEG₄-maleimide (DBCO-PEG₄-MAL; purity > 95%) and dibenzocyclooctyne-PEG₄-carboxyrhodamine 110 (DBCO-PEG₄-carboxyrhodamine; purity > 95%) from Click Chemistry Tools LLC (Scottsdale, AZ, USA). Disposable PD-10 desalting columns, HiTrap Q HP anion exchange columns, and HiTrap SP HP cation exchange columns were purchased from GE Healthcare (Little Chalfont, Buckinghamshire, UK). We purchased Vivaspin centrifugal concentrators (each with a molecular weight cut-off of 10 kDa) from Sartorius (Weender Landstraße, Göttingen, Germany). The GLP1_C peptide was synthesized by GenScript (Piscataway, NJ, USA). Anti-GLP-1 monoclonal antibody was purchased from Thermo Fisher Scientific (Waltham, MA, USA). Fetal bovine serum and antibiotic-antimycotic for cell culture were purchased from Gibco (Gaithersburg, MD, USA). X-tremeGENE HP DNA transfection reagent was purchased from Roche Diagnostics GmbH (Mannheim, Germany). cAMP Parameter Assay Kit was purchased from R&D Systems (Minneapolis, MN, USA). Iscove’s modified Dulbeco’s medium and all other chemicals (at least ACS reagent grade) were purchased from Sigma-Aldrich unless otherwise indicated.

Bak M., Park J., Min K., Cho J., Seong J., Hahn Y.S., Tae G, & Kwon I. (2020). Recombinant Peptide Production Platform Coupled with Site-Specific Albumin Conjugation Enables a Convenient Production of Long-Acting Therapeutic Peptide. Pharmaceutics, 12(4), 364.

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Oxidized DJ-1 Antibody Preparation

Cited 1 time

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Hydrogen peroxide (H₂O₂) and isopropyl-β-d-1-thiogalactopyranoside (IPTG) were purchased from Wako Pure Chemical Industries, Osaka, Japan; 3,3′,5,5′-tetramethylbenzidine (TMB), 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine hydrochloride (MPTP), and anti-β actin (AC-15) were obtained from Sigma-Aldrich, St. Louis, MO; nickel-nitrilotriacetic acid (Ni-NTA) agarose was obtained from Qiagen GmbH, Hilden, Germany; and protease inhibitor cocktail tablets were obtained from Nacalai Tesque, Kyoto, Japan. Monoclonal antibodies (mAbs) against oxDJ-1 were prepared as previously described30 (link)31 (link). In each experimental system for the analysis of oxDJ-1, the most suitable mAb clone was selected based on the specificity and sensitivity. Other chemicals used were of the highest quality commercially available.

Saito Y., Akazawa-Ogawa Y., Matsumura A., Saigoh K., Itoh S., Sutou K., Kobayashi M., Mita Y., Shichiri M., Hisahara S., Hara Y., Fujimura H., Takamatsu H., Hagihara Y., Yoshida Y., Hamakubo T., Kusunoki S., Shimohama S, & Noguchi N. (2016). Oxidation and interaction of DJ-1 with 20S proteasome in the erythrocytes of early stage Parkinson’s disease patients. Scientific Reports, 6, 30793.

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Engineered HEV ORF2 Capsid Mutants

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Plasmid pET-28a (+)/p179 containing the 439–617aa region of HEV ORF2 of genotype 4 HEV strain has been constructed previously in our laboratory^{14 (link),28 (link)}. The p179 mutants with wild-type asparagine (N) replaced by the cyclic aa (P) and aromatic aa (Y) at position 562 were previously designed and successfully expressed in P. pastoris^{14 (link)}. HEV capsid protein-specific mAb (1G10) was produced by our research group^{13 (link)}. Isopropyl-β-D-thiogalactopyranoside (IPTG), High-fidelity DNA polymerase, dNTP, T4 DNA ligase and restriction endonucleases were purchased from Roche (Germany). E. coli BL21(DE3) cells were purchased from Promega. HRP-conjugated goat anti-mouse was from KPL (Gaithersburg, MD, USA). Trypsin and DAB were purchased from Sigma–Aldrich (St. Louis, MO, USA). Plasmid and DNA recovery/purification kits were obtained from Axygen, Inc (USA). The nickel-nitrilotriacetic acid (Ni-NTA) Agarose was obtained from QIAGEN Sciences, MD, USA.

Wei W., Behloul N., Baha S., Liu Z., Aslam M.S, & Meng J. (2018). Dimerization: a structural feature for the protection of hepatitis E virus capsid protein against trypsinization. Scientific Reports, 8, 1738.

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Purification of His6-tagged SAS1 from E. coli

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Escherichia coli BL21 (DE3) harboring pET24d::his₆-SAS1 was grown at 37°C until it reached an OD₆₀₀ of ~0.6–0.8 and was induced by adding 12.5 g/l d(+)-lactose monohydrate (Sigma). After 20 h at 30°C, cells were harvested as described. The cell pellet was resuspended in 10 ml of wash buffer [20 mM HEPES/NaOH (pH 8.0), 250 mM NaCl, 40 mM imidazole, 20 mM MgCl₂, 20 mM KCl], supplemented 0.1 mg/ml of DNase I, RNase A, lysozyme, and 1 × cOmplete^™ EDTA-free protease inhibitor cocktail (Roche). Cells were lysed by three passages through a French press mini cell at 900 psi. The cleared lysate was applied to 1 ml of equilibrated nickel nitrilotriacetic acid (Ni-NTA) agarose (Qiagen) and washed three times with 10 ml of wash buffer. ^HisSAS1 was eluted by adding 1 ml of elution buffer [20 mM HEPES/NaOH (pH 8.0), 250 mM NaCl, 500 mM imidazole, 20 mM MgCl₂, 20 mM KCl] five times. ^HisSAS1 was buffer exchanged in storage buffer [20 mM HEPES/NaOH (pH 7.5), 200 mM NaCl, 20 mM MgCl₂, 20 mM KCl] and concentrated using a 30 kDa Amicon^® (Millipore). Protein concentration was determined using the Roti^®-Quant reagent (Carl Roth) using BSA as a standard, and the aliquots were stored at −80°C.

Brückner S., Müller F., Schadowski L., Kalle T., Weber S., Marino E.C., Kutscher B., Möller A.M., Adler S., Begerow D., Steinchen W., Bange G, & Narberhaus F. (2023). (p)ppGpp and moonlighting RNases influence the first step of lipopolysaccharide biosynthesis in Escherichia coli. microLife, 4, uqad031.

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Optimized Microbial Protein Production

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E. coli strains DH5α and BL21 (DE3), and the plasmid pET-22b and pET-28b are products of Novagen (USA). Human cDNA library was supplied by Maikun Teng, University of Science and Technology of China. The genes encoding the EmGFP, mCherry, the codon-optimized Vhb, the mature mSF (V71-S212) with insertion at BamH I and Xho I sites in the expression vectors, and the helper plasmid for coexpressing yeast mature 5-aminolevulinic acid synthase (ALAS) were constructed by our laboratory [24 (link), 25 (link)].
Heparin Sepharose CL-6B, Phenyl Sepharose CL-4B and amylose resin were obtained from GE (Healthcare, USA). Nickel-nitrilotriacetic acid (Ni–NTA) agarose was purchased from Qiagen (Hilden, Germany). Ultra-15 centrifugal filter tubes equipped with the Ultracel-10 membrane were purchased from Merck-Sigma-Aldrich (Kenilworth, NJ, USA). Primers and the gene encoding the CrLOV were synthesized in General Biol Company (Chuzhou, Anhui, China). Monoclonal antibody including mouse anti-GFP, and anti-MBP, and horseradish peroxidase (HRP) conjugated goat anti-mouse IgG antibody, reagents for Western blot analysis were bought from Transgen Biotech (Beijing, China). RAC was prepared from microcrystalline cellulose, according to our previous report [24 (link)].

He X., Zhang S., Dang D., Lin T., Ge Y., Chen X, & Fan J. (2023). Detection of human annexin A1 as the novel N-terminal tag for separation and purification handle. Microbial Cell Factories, 22, 2.

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Purification of C-terminal His-tagged H-NS

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H-NS wild type and variants with a 6× His tag at their C-terminus were expressed and purified using pHGE-based plasmids in S. oneidensis Δhns strain. Cells were induced for 6 h (at A₆₀₀ ∼ 0.6) with 0.5 mM IPTG and were then harvested and lysed at 30 kpsi by a cell disruptor (Constant Systems Ltd., Northants, UK) in lysis buffer (50 mM NaH₂PO₄, 300 mM NaCl, pH 8.0) with the addition of protease inhibitor cocktail (Sigma-Aldrich, St. Louis, MO, USA). Nickel-nitrilotriacetic acid (Ni-NTA) agarose (Qiagen, Valencia, CA, USA) was used to purify the His-tagged proteins following the manufacturer's instructions. The purified proteins were dialyzed into a buffer (20 mM Tris–HCl, 300 mM NaCl, pH 8.0) for further analysis and were quantified with a bicinchoninic acid (BCA) assay kit (Bio Teke, Beijing, China).

Liu X., Lin S., Liu T., Zhou Y., Wang W., Yao J., Guo Y., Tang K., Chen R., Benedik M.J, & Wang X. (2021). Xenogeneic silencing relies on temperature-dependent phosphorylation of the host H-NS protein in Shewanella. Nucleic Acids Research, 49(6), 3427-3440.

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