Ni nta affinity resin

Manufactured by Thermo Fisher Scientific

Sourced in United States, China

Ni-NTA affinity resin is a chromatographic material used for the purification of recombinant proteins. It consists of nickel-nitrilotriacetic acid (Ni-NTA) immobilized on agarose beads. The Ni-NTA resin selectively binds to proteins containing a polyhistidine (His-tag) sequence, allowing for the efficient capture and purification of target proteins from complex mixtures.

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Lab products found in correlation

Bca protein assay kit, by Thermo Fisher Scientific (4 mentions) Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions) Imidazole, by Merck Group (1 mentions) Pfastbachtb vector, by Thermo Fisher Scientific (1 mentions) Bradford s reagent, by Bio-Rad (1 mentions) Akta prime plus system, by GE Healthcare (1 mentions) Hiload 16 600 superdex 200 pg column, by GE Healthcare (1 mentions) Saccharose, by Merck Group (1 mentions) Isopropyl β d thiogalactopyranoside iptg, by Merck Group (1 mentions) Pet 32a, by Sangon (1 mentions)

Spelling variants of this product

Ni-NTA resin (147 citations) Ni-NTA (43 citations) Ni-NTA affinity (2 citations) His-Tag Ni-affinity resin (Ni-NTA Agarose, (2 citations) HisPur Ni-NTA affinity resin (2 citations)

8 protocols using ni nta affinity resin

Baculovirus-Expressed PI3Kγ Protein Purification

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Baculovirus construct was generated by subcloning wild type full length PI3Kγ downstream of 6× His tag sequence into the pFastBacHTb vector (Invitrogen) as previously described [10 (link)]. The cloned bacmid DNA was used to infect Sf9 insect cells to express recombinant proteins. Sf9 cells expressing PI3Kγ protein were collected and re-suspended in cell lysis buffer [HEPES pH 7.4, 50 mM KCl, 300 mM NaCl, 10% glycerol, 14 mM β-mercaptoethanol (β-ME), protease inhibitor cocktail]. The suspended cells were sonicated and incubated with Benzonase (5 U/mL) for 30 min and the suspension was centrifuged at 35,000 × g for 30 min. The PI3Kγ protein was immunoprecipitated using Ni-NTA affinity resin (Invitrogen) following manufacturer’s protocol. Wash buffer consisted of cell lysis buffer plus 5 mM imidazole (Sigma). The protein was eluted using elution buffer (Cell lysis buffer plus 150 mM imidazole). The immunoprecipitated PI3Kγ was used in in vitro protein kinase assays using Src as substrate and in vitro Src functional assays with enolase from baker’s yeast (Sigma) as substrate.

Watson L.J., Alexander K.M., Mohan M.L., Bowman A.L., Mangmool S., Xiao K., Naga Prasad S.V, & Rockman H.A. (2016). Phosphorylation of Src by phosphoinositide 3-kinase regulates beta-adrenergic receptor mediated EGFR transactivation. Cellular signalling, 28(10), 1580-1592.

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CCDC47 Immunoprecipitation Methods

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Unless otherwise indicated, all CCDC47 IPs were performed with CCDC47 antibody #1. For immunoprecipitations under denaturing conditions, samples of interest were first denatured in 1% SDS and 100 mM Tris pH 8.0 and boiled for 2-5 min. Samples were diluted 10-fold in IP buffer (100 mM NaCl, 50 mM HEPES pH 7.4, 1% TritonX-100) then immunoprecipitated in batch with desired antibodies at 4°C rotating end-over-end for 2-4 h. Unbound supernatant was removed by aspiration and beads were washed 3x in IP buffer before elution in 2x SDS- PAGE sample buffer. Native immunoprecipitation followed similar protocols but were performed in the presence of NSB (200 mM KOAc, 25 mM HEPES pH 7.4, 1% DBC). EDTA and RNase A was added prior to solubilisation where RNase treatment is indicated in the figure legends. Pulldowns of His-tagged β₁AR intermediates and full length β1AR were performed using Ni-NTA affinity resin (Invitrogen) in 1xPBS +250 mM NaCl, 0.5% TritonX-100, 10 mM Imidazole.

Chitwood P.J, & Hegde R.S. (2020). An intramembrane chaperone complex facilitates membrane protein biogenesis. Nature, 584(7822), 630-634.

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Neutralizing N. kaouthia Neurotoxin with HuScFv

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O. hannah holovenom and horse-derived monospecific N. kaouthia antivenin (purified equine F(ab)’2) were obtained in lyophilized form from the Queen Saovabah Memorial Institute (QSMI). The lyophilized antivenin was dissolved in ten mL of ultrapure sterile distilled water (UDW), while the venom was dissolved in one mL of normal saline solution (NSS). Protein concentrations of the preparations were determined by using Bradford’s reagent (Bio-Rad, Hercules, CA, USA).
For preparing NkLN-HuScFv, the gene sequence coding for the HuScFv (huscfv) in phagemid transformed E. coli clone no. P8/22/3 [10 (link)] was subcloned into pET23b⁺, and the recombinant plasmids were put into BL21 (DE3) E. coli. The transformed bacteria were grown under IPTG induction condition, and soluble NkLN-HuScFv was purified from the bacterial lysate by using Ni-NTA affinity resin (Thermoscience, Rockford, IL, USA). The E. coli-derived NkLN-HuScFv has been shown to neutralize N. kaouthia neurotoxin and rescued the N. kaouthia envenomed mice from lethality [10 (link)]. By using the phage peptide mimotope search and multiple alignments, the HuScFv was found to bind to amino acids in loop 3 of N. kaouthia long neurotoxin (accession No. 229777), which is the venom binding site to the neuronal acetylcholine receptor (AchR) [10 (link)].

Danpaiboon W., Reamtong O., Sookrung N., Seesuay W., Sakolvaree Y., Thanongsaksrikul J., Dong-din-on F., Srimanote P., Thueng-in K, & Chaicumpa W. (2014). Ophiophagus hannah Venom: Proteome, Components Bound by Naja kaouthia Antivenin and Neutralization by N. kaouthia Neurotoxin-Specific Human ScFv. Toxins, 6(5), 1526-1558.

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Purification of the Rpn10 Proteasome Subunit

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Purification of Rpn10 was conducted as previously described (Lander et al., 2012 (link)). Briefly, Bl21-star (DE3) E. coli cells were transformed with pACYCDuet-1 containing 6xHis-HRV-Rpn10. The cells were grown in dYT media at 37 °C until OD600 = 0.6-0.8. Protein expression was induced with 0.5 mM IPTG at 37 °C for 4 hr. After induction, the cells were harvested and resuspended in lysis buffer (60 mM HEPES, pH 7.6, 100 mM NaCl, 100 mM KCl, 20 mM Imidazole, 10% glycerol) containing 2 mg/mL lysozyme, benzonase, and protease inhibitors (aprotinin, pepstatin, leupeptin and PMSF). The cells were lysed by sonication, clarified by centrifugation, and the clarified lysate was bound in batch to 5 ml of Ni-NTA affinity resin (ThermoFisher). The resin was washed with lysis buffer and eluted with lysis buffer plus 250 mM imidazole. 2 mM DTT was added directly to the elution and the protein was cleaved overnight with HRV-protease. The next day the protein was purified using a Superdex 75 16/60 column equilibrated with lysis buffer.

Bard J.A., Bashore C., Dong K.C, & Martin A. (2019). The 26S Proteasome utilizes a kinetic gateway to prioritize substrate degradation. Cell, 177(2), 286-298.e15.

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Purification of Ebola VP40 Dimer

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Full length 6xHis-VP40 in pET46 was transformed into Rosetta BL21 DE3 cells (Novagen, Madison, Wisconsin). One colony was picked from the transformation plate and grown at 37°C at 200 rpm until an OD₆₀₀ of 0.4–0.6. VP40 expression was induced with 1 mM IPTG for 5 hours at room temperature. Protein was purified using a Ni NTA affinity resin (Thermo Scientific, Waltham, MA). Eluted protein was further purified using size exclusion chromatography (HiLoad 16/600 Superdex 200 pg column on an AKTA Prime Plus system (GE Healthcare Life Sciences, Pittsburgh, PA)) to separate the VP40 dimer and oligomer. VP40 dimer protein was concentrated and stored in 10 mM Tris, pH 8.0 containing 300 mM NaCl. Protein concentration was determined with a Pierce BCA Protein Assay Kit according to the manufacturer’s instructions (Thermo Scientific).

GC J.B., Pokhrel R., Bhattarai N., Johnson K.A., Gerstman B.S., Stahelin R.V, & Chapagain P.P. (2017). Graphene-VP40 interactions and potential disruption of the Ebola virus matrix filaments. Biochemical and biophysical research communications, 493(1), 176-181.

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Recombinant Fusion Protein BLP Purification

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A fusion gene BLP was generated by concatenating the oligonucleotides encoding a duplicated Lys-Asn-Pro-Tyr peptide, a linker (GGGG), and albumin-binding peptide [27 (link)]. The gene was amplified by polymerase chain reaction (PCR) using primers obtained from Sangon Co. Ltd. (Shanghai, China). The PCR conditions were 30 cycles of 30 s at 95 °C, 30 s at 55 °C, and 1 min at 72 °C. The amplified gene was cloned into the prokaryotic expression vector pET-32a(+) (Sangon, Shanghai, China) and the resulting BLP expression plasmids were transformed into E. coli strain BL21 (DE3) (Stratagene, La Jolla, CA, USA). After induction with 24 μg/mL isopropyl β-D-thiogalactopyrano-side (IPTG; Sigma, St. Louis, MO, USA) and 0.01 g/mL saccharose (Sigma, St. Louis, MO, USA) for 20 h at 24.5 °C, the BLP fusion conjugate was purified using Ni-NTA affinity resin (ThermoFisher, Shanghai, China). The recombinant bacterial protein was separated on a 12% (v/v) SDS-PAGE gel and a 28 kDa band corresponding to the recombinant hybrid polypeptide, including the vector fusion protein (20 kDa) and the hybrid polypeptide (approx. 8 kDa), was isolated. Protein concentration was determined with a bicinchoninic acid (BCA) assay.

Zeng Y., Gong Z., Wu B., Guan W., Yu S., An Y., Lu R., Zhao J., Wu Y., Huang Y, & Wu X. (2019). A novel Bursin-like peptide as a potential virus inhibitor and immunity regulator in SPF chickens infected with recombinant ALV. BMC Veterinary Research, 15, 447.

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Plasmid-based Protein Expression and Purification

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Plasmids encoding cDNA were obtained from Jiahuai Han's Lab: YTHDF2 (13328) and DDX1 (7395). Sequences were cloned into pET28a vector for protein expression (YTHDF2: EcoR I, Hand III; DDX1: BamHI, XhoI) (See Table S1 for primers cloning pET28a-YTHDF2, pET28a-DDX1 and DDX1 truncations). YTHDF2-YTH (396-580) was expressed for 20 hours at 16°C with 0.8 mM IPTG in Escherichia coli strain BL21 (DE3), DDX1 and truncations was expressed for 16 hours at 18°C with 0.5 mM IPTG in Escherichia coli strain Rosetta (DE3). Cells were lysed by sonication and the protein was purified using Ni-NTA affinity resin (20349, Thermo Fisher Scientific) according to the manufacturer's recommendations. The purity of recombinant proteins exceeded 90% as detected SDS-PAGE analysis (Figure S1). Protein concentrations were determined by BCA Protein Assay Kit (23227, Thermo Fisher Scientific) and characterized by LC-MS/MS. Mass spectrometric data of the recombinant proteins are shown in Figure S1.

Huang Y., Bai X., Guo Z., Dong H., Fu Y., Zhang H., Zhai G., Tian S., Wang Y, & Zhang K. (2021). DNA-guided photoactivatable probe-based chemical proteomics reveals the reader protein of mRNA methylation. iScience, 24(9), 103046.

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Recombinant YTHDF2 and DDX1 Protein Production

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Plasmids encoding cDNA were obtained from Jiahuai Han's Lab: YTHDF2 (13328) and DDX1 (7395). Sequences were cloned into pET28a vector for protein expression (YTHDF2: EcoR I, Hand III; DDX1: BamHI, XhoI) (See Table S1 for primers cloning pET28a-YTHDF2, pET28a-DDX1 and DDX1 truncations). YTHDF2-YTH (396-580) was expressed for 20 hours at 16 C with 0.8 mM IPTG in Escherichia coli strain BL21 (DE3), DDX1 and truncations was expressed for 16 hours at 18 C with 0.5 mM IPTG in Escherichia coli strain Rosetta (DE3). Cells were lysed by sonication and the protein was purified using Ni-NTA affinity resin (20349, Thermo Fisher Scientific) according to the manufacturer's recommendations. The purity of recombinant proteins exceeded 90% as detected SDS-PAGE analysis (Figure S1). Protein concentrations were determined by BCA Protein Assay Kit (23227, Thermo Fisher Scientific) and characterized by LC-MS/MS. Mass spectrometric data of the recombinant proteins are shown in Figure S1.

Huang Y., Bai X., Guo Z., Dong H., Zhai G., Zhang H., Tian S., Wang Y., & Zhang K. (2020). DNA-Guided Photoactivatable Probe-Based Chemical Proteomics Reveals the Reader Protein of mRNA Methylation.

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