Ni2 nitrilotriacetic acid resin

Manufactured by Qiagen

Sourced in Germany

Ni2+-nitrilotriacetic acid resin is a chromatography resin used for the purification of recombinant proteins containing a polyhistidine tag. The resin utilizes the high affinity interaction between the nickel ions (Ni2+) and the polyhistidine tag to selectively bind and capture the target protein from complex mixtures.

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Lab products found in correlation

Gsh sepharose, by GE Healthcare (2 mentions) Thrombin, by Solarbio (1 mentions) Superose 6 column, by GE Healthcare (1 mentions) Source 15q column, by GE Healthcare (1 mentions) Anti β actin ac 15, by Merck Group (1 mentions) Conalbumin, by GE Healthcare (1 mentions) Ovalbumin (ova), by GE Healthcare (1 mentions) Superdex 75 column, by GE Healthcare (1 mentions)

8 protocols using ni2 nitrilotriacetic acid resin

Recombinant Tyrosine Hydroxylase Protein Purification

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Recombinant proteins with His or GST tag were expressed in Rosetta (DE3) pLys Escherichia coli (Novagen, Bilerica, MA). Cultures were grown until linear phase (0.6 OD) and expression of proteins were induced by 250 μM IPTG at 18 °C for 20 h. As iron is essential for TH activity, to maintain TH activity 2% glucose and 1 mM FeSO₄ together were added when inducing the expression of recombinant TH as reported29 (link). The culture was then harvested and recombinant proteins were purified by Ni²⁺-nitrilotriacetic acid resin (Qiagen) or GSH-Sepharose (GE Healthcare, Piscataway, NJ) according to the manufacturer’s protocol, respectively. The obtained proteins were then dialyzed in PBS.
For the generation of anti-TH antibody, purified his-tagged TH recombinant protein in PBS was mixed with equal volume of Freund’s adjuvant forming an emulsion. The mixture was then used to immunize a New Zealand white rabbit and the Anti-TH antibody was purified from serum with GST-tagged TH conjugated to CNBr activated Sepharose affinity purification (GE Healthcare, Piscataway, NJ). The specificity of the antibodies was verified by Western blot analysis.

Wang Y., Sung C.C, & Chung K.K. (2017). Novel enhancement mechanism of tyrosine hydroxylase enzymatic activity by nitric oxide through S-nitrosylation. Scientific Reports, 7, 44154.

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Recombinant RhgmaS Protein Production

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The full-length RhgmaS gene from R. sp. 12E13 was synthesized by the Beijing Genomics Institute (China). The gene was then cloned into the pET-28a vector containing an N-terminal His-tag (Novagen). All point mutations of RhGmaS were created by the PCR-based method and confirmed by DNA sequencing. The RhGmaS protein and all the mutants were expressed in E. coli BL21 (DE3). The recombinant strains were grown at 37 °C in the lysogeny broth medium containing 30 μg/ml kanamycin and then were induced by 0.5-mM IPTG at 18 °C for 16 h. All proteins were purified first by Ni²⁺ nitrilotriacetic acid resin (Qiagen, Germany) and then by anion-exchange chromatography on a Source 15Q column (GE Healthcare) with 0- to 1-M NaCl in 50-mM Tris-HCl (pH 8.0). The eluted proteins were further fractionated by gel filtration on a Superose 6 column (GE Healthcare) with the buffer containing 10-mM Tris HCl (pH 8.0) and 100-mM NaCl. To prepare RhGmaS protein without His-tag, we digested the purified RhGmaS by thrombin (Solarbio, China) at 4 °C overnight, and then the mixtures were fractionated by gel filtration on a Superose 6 column.

Wang N., Chen X.L., Gao C., Peng M., Wang P., Zhang N., Li F., Yang G.P., Shen Q.T., Li S., Chen Y., Zhang Y.Z, & Li C.Y. (2020). Crystal structures of γ-glutamylmethylamide synthetase provide insight into bacterial metabolism of oceanic monomethylamine. The Journal of Biological Chemistry, 296, 100081.

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Purification and Antibody Generation for NME7

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Recombinant proteins containing a hexahistidine (His₆) or glutathione S-transferase (GST) tag were expressed in Escherichia coli BL21 (DE3) and then isolated using Ni²⁺-nitrilotriacetic acid resin (Qiagen, Valencia, CA) or GSH-Sepharose (GE Healthcare, Chalfont St. Giles, Buckinghamshire, United Kingdom), respectively. After elution, the proteins were dialyzed in 50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM ethylene glycol tetraacetic acid (EGTA), and 10% glycerol and then stored at −80°C.
Two anti-NME7 antisera were generated by immunizing rabbits with NME7 1–140 and full-length NME7 prepared as His₆-tagged proteins. The sera obtained were named sera 651 and 673, respectively. Both antibodies were purified using respective antigens in fusion with GST and immobilized on polyvinylidene fluoride membranes. The two antibodies gave similar results in experiments, and most of the data presented here were collected using antibody 651. The production of the following antibodies has been described previously: rabbit anti-CDK5RAP2, anti-GCP2, anti-GCP3, anti-GCP4, anti-GCP5, and anti-GCP6 (Fong et al., 2008 (link); Choi et al., 2010 (link)). These mouse monoclonal antibodies were purchased from Sigma (St. Louis, MO): anti-FLAG (M2), anti–γ-tubulin (GTU-88), anti–α-tubulin (DM1A), and anti–β-actin (AC-15).

Liu P., Choi Y.K, & Qi R.Z. (2014). NME7 is a functional component of the γ-tubulin ring complex. Molecular Biology of the Cell, 25(13), 2017-2025.

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Purification of STIM1 CC3ext Protein

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STIM1 CC3ext (i.e. residues 388–491) was subcloned into pET-28a using NheI and XhoI restriction sites. Protein was expressed in BL21 ΔE3 Escherichia coli, and Ni²⁺-nitrilotriacetic acid resin (Qiagen) was used to pull out the 6×His-tagged CC3ext from guanidinium-solubilized lysate. Refolding was performed by dilution in 20 mm Tris, 200 mm NaCl, 0.8 mm DTT, 0.8 mm EDTA, pH 8.5, and the protein was further purified by gel filtration after thrombin cleavage (10 units/mg overnight at 4 °C) of the 6×His affinity tag. Protein homogeneity was assessed by Coomassie-stained SDS-PAGE.

Fahrner M., Muik M., Schindl R., Butorac C., Stathopulos P., Zheng L., Jardin I., Ikura M, & Romanin C. (2014). A Coiled-coil Clamp Controls Both Conformation and Clustering of Stromal Interaction Molecule 1 (STIM1). The Journal of Biological Chemistry, 289(48), 33231-33244.

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Purification and Storage of GAS2L1 Proteins

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Recombinant GAS2L1 proteins containing a His₆ or His₆-FLAG tag were expressed in Escherichia coli BL21 (DE3), purified using Ni²⁺-nitrilotriacetic acid resin (Qiagen), and dialyzed in PBS containing 10% glycerol. Proteins used in F-actin sedimentation assays were dialyzed in G-buffer (5 mM Tris-HCl, pH 8.0, and 0.2 mM CaCl₂). After dialysis, the proteins were stored in aliquots at −80°C.

Au F.K., Hau B.K, & Qi R.Z. (2020). Nek2-mediated GAS2L1 phosphorylation and centrosome-linker disassembly induce centrosome disjunction. The Journal of Cell Biology, 219(5), e201909094.

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Expression and Purification of UrtA Protein

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The wildtype UrtA and its mutants were expressed in E. coli strain BL21 (DE3) (Vazyme). Cells were cultured at 37 °C in HB-PET autoinduction medium (Haibo) to an absorbance at 600 nm (A₆₀₀) of 0.8 to 1.0 and then cultured at 18 °C for 32 h. Then, the cells were collected and lysed in the buffer containing 40 mM Tris-HCl (pH 8.0), 0.2 M NaCl, 5% (v/v) glycerol, 2 mM ethylene diamine tetraacetic acid, and 0.1 mM phenylmethanesulfonyl fluoride. The proteins were purified first with Ni²⁺-nitrilotriacetic acid resin (Qiagen) and then fractionated by gel filtration on a Superdex-75 column (GE Healthcare) in the buffer containing 20 mM Tris-HCl (pH 8.0), 200 mM NaCl, and 1% (v/v) glycerol. Ovalbumin (43 kDa) and conalbumin (75 kDa) from GE Healthcare were used as protein size standards to analyze the aggregative form of UrtA.

Wang C., Zhu W.J., Ding H.T., Liu N.H., Cao H.Y., Suo C.L., Liu Z.K., Zhang Y., Sun M.L., Fu H.H., Li C.Y., Chen X.L., Zhang Y.Z, & Wang P. (2023). Structural and molecular basis for urea recognition by Prochlorococcus. The Journal of Biological Chemistry, 299(8), 104958.

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Purification and Labeling of Biomolecules

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Various chemicals and reagents were obtained from E. Merck (Mumbai, India) Limited; Sisco Research Laboratories Pvt. Ltd., Mumbai, India; HiMedia Laboratories Limited (Mumbai, India) and from Sigma Chemical Co., St. Louis, MO, USA. The Ni⁺²‐nitrilotriacetic acid resin (QIAGEN GmbH, Hilden, Germany), dNTP mix (Fermentas Life Sciences, Waltham, MA, USA), Immobilon‐P (PVDF, polyvinylidene fluoride) membrane (Millipore, Billerica, MA, USA) and [γ³²‐P] GTP (specific activity 4500 Ci·mmol⁻¹) (BRIT, Mumbai, India) were purchased from the indicated sources.
Cellulose thin layer (100 μm) chromatography plates (polyester sigmacell type 100 cellulose) of dimensions 20 cm × 20 cm, were purchased from Sigma.

Ghosh A., Dutta D., Bandyopadhyay K, & Parrack P. (2016). Characterization of the autophosphorylation property of HflX, a ribosome‐binding GTPase from Escherichia coli. FEBS Open Bio, 6(7), 651-659.

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Purification of Prefusion RSV F Protein

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RSV F mutations were carried out by site-directed mutagenesis using Quik-Change (Stratagene), as was the addition of the AviTag sequence at the C terminus of the RSV F protein. Expi293F cells were transiently transfected with plasmids expressing RSV F site Ø and site II KO pre-F constructs and grown in suspension. The culture supernatants were harvested 5 days after transfection and centrifuged at 10,000 rpm to remove cell debris. The culture supernatants were sterile-filtered, and RSV F glycoproteins were purified by a Ni²⁺–nitrilotriacetic acid resin (Qiagen) and a Strep-Tactin resin (Novagen). Relevant fractions containing the RSV F variants were pooled, concentrated, and subjected to size-exclusion chromatography. Fractions corresponding to the trimer peak (fig. S5) were concentrated, and frozen at −80°C.

Ngwuta J.O., Chen M., Modjarrad K., Joyce M.G., Kanekiyo M., Kumar A., Yassine H.M., Moin S.M., Killikelly A.M., Chuang G.Y., Druz A., Georgiev I.S., Rundlet E.J., Sastry M., Stewart-Jones G.B., Yang Y., Zhang B., Nason M.C., Capella C., Peeples M.E., Ledgerwood J.E., McLellan J.S., Kwong P.D, & Graham B.S. (2015). Prefusion F–specific antibodies determine the magnitude of RSV neutralizing activity in human sera. Science translational medicine, 7(309), 309ra162.

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