Ni nta agarose resin

Manufactured by Thermo Fisher Scientific

Sourced in United States

Ni-NTA agarose resin is a chromatography medium used for the purification of histidine-tagged recombinant proteins. It is composed of nickel-nitrilotriacetic acid (Ni-NTA) coupled to agarose beads, which allows for the selective binding and purification of proteins containing polyhistidine tags.

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Lab products found in correlation

Pd 10 column, by GE Healthcare (4 mentions) 3h l glutamine, by PerkinElmer (3 mentions) Zeocin, by Thermo Fisher Scientific (3 mentions) Ppiczb vector, by Thermo Fisher Scientific (3 mentions) Expi293 cell, by Thermo Fisher Scientific (2 mentions) Amicon ultra 4, by Merck Group (2 mentions) Hybond ecl membrane, by GE Healthcare (2 mentions) Ecl plus, by GE Healthcare (2 mentions) Hybond, by Cytiva (2 mentions) L glutamine, by Merck Group (2 mentions) Gibson assembly cloning, by New England Biolabs (1 mentions) Superdex 75 10 300 column, by GE Healthcare (1 mentions) Hispur cobalt resin, by Thermo Fisher Scientific (1 mentions) Chemidoc mp imaging system, by Bio-Rad (1 mentions) Isopropyl β d 1 thiogalactopyranoside (iptg), by Roche (1 mentions) Glutathione agarose resin, by Thermo Fisher Scientific (1 mentions) Ktapurifier upc 10, by GE Healthcare (1 mentions) Anti flag m2 resin, by Merck Group (1 mentions) Flag peptide, by Merck Group (1 mentions) Superdex 75 column, by Cytiva (1 mentions)

Spelling variants of this product

Agarose (403 citations) Ni-NTA resin (147 citations) Ni-NTA agarose (143 citations) Ni-NTA (43 citations) HisPur Ni-NTA agarose resin (6 citations) His-Tag Ni-affinity resin (Ni-NTA Agarose, (2 citations) Nickel-nitrilotriacetic acid (Ni-NTA) agarose resin (2 citations) HisPur™ Nickel-nitrilotriacetic acid-agarose (Ni-NTA) resin (2 citations)

31 protocols using ni nta agarose resin

Purification of SARS-CoV-2 Variant Antigens

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Genes encoding RBD of SARS-CoV-2 Alpha, Beta, Gamma, Delta, and Omicron variants were cloned in-frame into the pcDNA3.4-SARS-CoV-2-spike RBD-his using Gibson Assembly cloning (NEB, Ipswich, MA, USA) [13 (link)]. SARS-CoV-2 antigens were produced in Expi293 cells (Thermo Fisher Scientific) and his–tagged SARS-CoV-2 antigens protein was purified using Ni-NTA agarose resin (Thermo Fisher Scientific) affinity chromatography, as described previously [13 (link)].
After 5 days of transfection, the supernatant was collected and passed over the Ni-NTA agarose resin column three times. First, the column was washed with 100 mL of 1× PBS to remove nonspecific bound proteins. Then, 3 mL of elution buffer (pH8.0, 50 mM sodium phosphate, 300 mM NaCl, and 250 mM imidazole) was added to elute the bound proteins. Finally, samples were buffer-exchanged into pH 7.4 PBS using Amicon Ultra-4 (Merck Millipore, Burlington, MA, USA) spin columns with a 10 kDa cutoff. The purity of purified samples was assessed by 14% SDS-PAGE gel (Additional file 1: Supplementary Fig. 1).

Kang C.K., Shin H.M., Choe P.G., Park J., Hong J., Seo J.S., Lee Y.H., Chang E., Kim N.J., Kim M., Kim Y.W., Kim H.R., Lee C.H., Seo J.Y., Park W.B, & Oh M.D. (2022). Broad humoral and cellular immunity elicited by one-dose mRNA vaccination 18 months after SARS-CoV-2 infection. BMC Medicine, 20, 181.

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Purification of Fn3 Variants for Binding Studies

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MSLN-binding Fn3 variant 3.4.4 and negative control protein Fn3 RDG, in which the RGD integrin-binding motif has been mutated to RDG (plasmid DNA provided by B. Hackel, University of Minnesota), were prepared as previously described (Sirois et al., 2018 (link)). Briefly, Fn3 genes were cloned into a pET vector with a C-terminal hexahistadine tag (plasmid provided by B. Hackel, University of Minnesota) and expressed in BL21(DE3) E. coli. Cultures were grown in LB and induced overnight at 20°C with 0.5 mM Isopropyl-β-D-thiogalactopyranoside (IPTG). Cells were resuspended in lysis buffer (35 mM Na₂HPO₄·dibasic, 15 mM NaH₂PO₄·monobasic, 500 mM NaCl, 5 mM CHAPS, 25 mM imidazole, 5% glycerol) supplemented with an EDTA-free protease inhibitor (Pierce), and lysed by repeated freezing and thawing. Soluble fractions were isolated by centrifugation. Fn3 variant 3.4.4 was purified by cobalt affinity chromatography with HisPur cobalt resin (Thermo Fisher) while Fn3 RDG was purified by nickel affinity chromatography with Ni-NTA agarose resin (Thermo Fisher) followed by size exclusion chromatography (SEC) on a Superdex 75 10/300 column (GE Healthcare Life Sciences). Protein samples were analyzed for purity by SDS-PAGE on a BioRad ChemiDoc MP imaging system.

Sirois A.R., Deny D.A., Li Y., Fall Y.D, & Moore S.J. (2019). Engineered Fn3 protein has targeted therapeutic effect on mesothelin-expressing cancer cells and increases tumor cell sensitivity to chemotherapy. Biotechnology and bioengineering, 117(2), 330-341.

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Purification of LOTUS and Vasa Proteins

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cDNA fragments of LOTUS domains were obtained by PCR amplification or gene synthesis (GenScript) and subcloned into pET28a (His-tag) vector (Supplementary Table S2). Drosophila Vasa (200–661aa) cDNA was obtained by PCR amplification and were subcloned into pGEX-4t-1 (GST-tag) vector. Plasmids were transformed into Escherichia coli (BL21 or Rosetta) and recombinant proteins were induced with 0.2 mM IPTG (Roche) at 18°C overnight. His-tagged proteins were affinity purified with Ni-NTA Agarose Resin (Thermo Scientific). GST-tagged proteins were affinity purified using Glutathione Agarose Resin (Thermo Scientific). Proteins were further purified by gel filtration chromatography with ÄKTApurifier UPC 10 (GE Healthcare). The running buffer for gel filtration chromatography is 10 mM Tris–HCl (pH 7.4) and 100 mM KCl.

Ding D., Wei C., Dong K., Liu J., Stanton A., Xu C., Min J., Hu J, & Chen C. (2020). LOTUS domain is a novel class of G-rich and G-quadruplex RNA binding domain. Nucleic Acids Research, 48(16), 9262-9272.

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SARS-CoV-2 Spike Protein Expression

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pcDNA3.1 SARS-CoV-2 S D614G was a gift from Jeremy Luban (Addgene plasmid # 158075; http://n2t.net/addgene:158075; RRID: Addgene_158075). SARS-CoV-2 S D614G protein, RBD_wt, RBD_δ, and RBD_ο were produced in Expi293 cells (Thermo Fisher Scientific) and were purified using Ni-NTA agarose resin (Thermo Fisher Scientific) affinity chromatography, as described previously (24 (link), 25 (link)).
Briefly, Expi293 cells were cultured at 37 °C with 5% CO₂ for five days after transfection of each plasmid encoding SARS-CoV-2 S D614G protein, RBD_wt, RBD_δ, or RBD_ο (BA1). The supernatant was collected and passed over the Ni-NTA agarose resin column three times. After washing with 100 mL of phosphate-buffered saline (PBS), the his-tagged protein was eluted by elution buffer (pH8.0, 50 mM sodium phosphate, 300 mM NaCl, and 250 mM imidazole). Finally, samples were buffer-exchanged into pH 7.4 PBS using Amicon Ultra-4 (Merck Millipore, Burlington, MA, USA) spin columns with a 10 kDa cutoff. The purity of purified samples was assessed by 14% SDS-PAGE gel.

Kim J., Jeong J., Lee C.M., Lee D.W., Kang C.K., Choe P.G., Kim N.J., Oh M.D., Lee C.H., Park W.B., Lee K.H, & Im S.A. (2022). Prospective longitudinal analysis of antibody response after standard and booster doses of SARS-COV2 vaccination in patients with early breast cancer. Frontiers in Immunology, 13, 1028102.

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Affinity Purification of Tagged REIIBP

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Around 5×10⁸ H929 cells were transduced with tagged REIIBP (H929::REIIBP) or H929 untouched used as control, and then harvested in 50 mM NaH₂PO₄ pH 7.4; 50 mM NaCl; 0.05% Tween; protease inhibitor (ROCHE). Cells were lysed by 3 cycles of freeze/thawing on dry ice and passed through a 10 gauge needle. Lysates were cleared by passing through 0.45 µm filter, spinning at 13000 rpm for 10 minutes and finally by incubating with 1 mL Agarose resin (Thermo Scientific). Equal amounts of protein from sample and control cleared lysates were loaded into chromatography columns filled with M2 anti-FLAG resin (SIGMA) and agitated at 4°C for 1 hour. Columns were washed with 50 mM NaH₂PO₄ pH 7.4; 300 mM NaCl; 0.05% Tween; protease inhibitor. Immuno complexes were eluted with 100 µg/ml 3×FLAG peptide (SIGMA). Eluates from the anti-Flag column were incubated for 1 hour in NiNTA Agarose resin (Thermo Scientific) and washed with 50 mM NaH2PO4 pH 7.4; 300 mM NaCl; 20 mM Imidazole; 0.05% Tween; protease inhibitor. NiNTA columns were eluted with 250 mM Imidazole.

Mirabella F., Murison A., Aronson L.I., Wardell C.P., Thompson A.J., Hanrahan S.J., Fok J.H., Pawlyn C., Kaiser M.F., Walker B.A., Davies F.E, & Morgan G.J. (2014). A Novel Functional Role for MMSET in RNA Processing Based on the Link Between the REIIBP Isoform and Its Interaction with the SMN Complex. PLoS ONE, 9(6), e99493.

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Purification of Recombinant Proteins

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Designs were cloned into pET-22b expression vectors using Gibson assembly. The correct cloning of the gene was verified by Sanger sequencing. Small-scale expression was performed in Escherichia coli BL21(DE3) cells grown in 200 ml cultures using auto-induction medium grown for 24 h at 25°C. Cells were lysed in 50 mM Tris–HCl pH 8.0, 250 mM NaCl supplemented with 5 mM β-mercaptoethanol and EDTA-free protease-inhibitor tablets (Thermo Fisher Scientific) using an Emulsiflex C3 homogenizer and affinity purified using Ni–NTA agarose resin (Thermo Fisher Scientific) in a gravity-flow column. Protein was washed with lysis buffer plus 100 mM imidazole and eluted in lysis buffer plus 500 mM imidazole. Eluted protein was dialyzed against imidazole overnight at 4°C. Samples were run on SDS–PAGE to assess purity before size-exclusion chromatography (SEC) using a Superdex 75 column (Cytiva Life Sciences) attached to an ÄKTA FPLC (Cytiva Life Sciences). Sodium azide was then added to SEC elution fractions at a concentration of 0.05% as well as EDTA at a concentration of 5 mM.

Miller J.E., Agdanowski M.P., Dolinsky J.L., Sawaya M.R., Cascio D., Rodriguez J.A, & Yeates T.O. (2024). AlphaFold-assisted structure determination of a bacterial protein of unknown function using X-ray and electron crystallography. Acta Crystallographica. Section D, Structural Biology, 80(Pt 4), 270-278.

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In vitro Rtn4 ubiquitination assay

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In vitro Rtn4 ubiquitination assays were described previously.^{15 (link)} For linking recombinant SdeC derivatives to agarose beads, Ni–NTA agarose resin (Thermo Fisher) was washed 3× with 1× ART buffer (20 mM Tris, 10 mM NaCl, pH 7.4) and mixed with His₆-SdeC followed by incubation for 1 h at 4 °C. Next, the SdeC-bound agarose resin was washed 3× with 1× ART buffer before processing to the two-step bead assay. During preincubation, SdeC-bound agarose beads were added to 10 mM Ub, and 100 mM nicotinamide 1, N6-ethenoadenine dinucleotide (ε-NAD, Sigma) in 1× ART buffer (SdeC final concentration 50 nM), and incubated for 1 h at 37 °C. After incubation, the beads were removed from reaction by spin filter columns. The supernatant containing modified Ub and excess ε-NAD were mixed with 400 nM GST-HA-Rtn4 and fresh unbound SdeC variants, then incubated for another hour at 37 °C. Reactions were terminated by addition of reducing loading buffer and boiling.

Zhang M., McEwen J.M., Sjoblom N.M., Kotewicz K.M., Isberg R.R, & Scheck R.A. (2021). Members of the Legionella pneumophila Sde family target tyrosine residues for phosphoribosyl-linked ubiquitination. RSC Chemical Biology, 2(5), 1509-1519.

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SdeC-Mediated Deubiquitination of Ub Tetramers

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For modification of Ub, 100 μg of K63-linked Ub tetramer (R&D Systems, Cat# UCB-310 or South Bay Bio, SBB-UP0073) or monomeric Ub (R&D Systems, Cat# U-100H) were incubated together with 100 μM β-NAD and 20 nM SdeC variants (WT, C118S/DUB⁻, E859A/ART⁻ and H416A/PDE⁻) in 250 μl of 1X ART buffer at 37°C for 1 h. For DUB inhibitor pretreatment, 1 μM ubiquitin-propargylamide (R&D Systems, Cat# U-214) was incubated with 100 nM SdeC variants at room temperature for 30 min before adding to Ub₄ (final concentration, 20 nM SdeC in 1X ART buffer). To remove the SdeC, 10 μl of a 50% slurry of Ni-NTA agarose resin (Thermo Fisher Scientific, Cat# 88221) was washed three times with 1X ART buffer, then added to the Ub-SdeC reaction for another 1hr with end-over-end rotation at 37°C. For removal of SdeC enzyme, the samples were centrifuged at 1000 × g for 2 min in a microcentrifuge spin filter column. The supernatant was then desalted using a NAP-5 column (Thermo Fisher Scientific, Cat# 45–000-151) and lyophilized with a Labconco Freezone12. Samples were resuspended in Ultrapure water to a final protein concentration of 1 mg/ml.

Kotewicz K.M., Zhang M., Kim S., Martin M.S., Chowdhury A.R., Tai A., Scheck R.A, & Isberg R.R. (2024). Sde Proteins Coordinate Ubiquitin Utilization and Phosphoribosylation to Promote Establishment and Maintenance of the Legionella Replication Vacuole. bioRxiv.

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Recombinant Human PARP1 and GCSF Purification

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The bacterial expression and purification of human PARP1 were carried out by following a previously published protocol.^{9 (link)} The purified protein was further passed through an acrodisc unit with mustang E membrane (Pall Corporation, Port Washington, NY) by following the manufacturer’s instructions. The final endotoxin levels (< 0.5 EU mg⁻¹ mL⁻¹) were measured using Pierce LAL chromogenic endotoxin quantitation kits (Thermo Fisher Scientific). Purified PARP1 was analyzed by SDS-PAGE, flash frozen using liquid nitrogen, and stored at −80°C.
Recombinant human GCSF was expressed through transient transfection into Expi293F cells using the polyethylenimine max (PEI-MAX) transfection regent. Culture media of Expi293F cells transfected with the expression construct were collected at days 3 and 6 post-transfection and centrifuged at 4,000×g for 30 min. The supernatant was dialyzed in PBS buffer for overnight and another 6 hours in PBS at 4 °C and then loaded on a gravity flow column packed with 2 mL of Ni-NTA agarose resin (Thermo Fisher Scientific), followed by washing with PBS containing 20 mM imidazole and eluting with PBS containing 400 mM imidazole. GCSF protein was then dialyzed in PBS buffer for overnight and another 6 hours in PBS at 4 °C and concentrated using a 10 kDa-cutoff amicon centrifugal concentrator. Purified human GCSF was analyzed by SDS-PAGE and stored at −80°C.

Cheng Q., Zhang X.N., Zhang L., Chen J., Wang Y, & Zhang Y. (2022). A Poly-ADP-Ribose Polymer-GCSF Conjugate. Biomacromolecules, 23(12), 5267-5272.

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Purification of RSV L-P Protein Complex

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A codon-optimized version of the RSV (strain A2) L protein ORF was expressed in insect cells as previously described (19 (link)). L-P protein complexes were isolated from cell lysates by affinity chromatography on Ni-NTA agarose resin (ThermoFisher). The resin was washed three times with 60 mM imidazole, two times with 100 mM imidazole, and the L-P complex was either eluted using TEV protease, or with 250 mM imidazole. The L-P preparation was then subjected to dialysis. Isolated L-P complexes were analyzed by SDS-PAGE and PageBlue staining (Fermentas) and the L protein concentration was estimated against bovine serum albumin reference standards. Experiments shown in Figures 1, 4, 5 and Supplementary Figure S1 were performed with both types of L-P preparations (no difference in experimental results between preparations was observed). Remaining experiments were performed with the TEV eluted L-P preparations.

Cressey T.N., Noton S.L., Nagendra K., Braun M.R, & Fearns R. (2018). Mechanism for de novo initiation at two sites in the respiratory syncytial virus promoter. Nucleic Acids Research, 46(13), 6785-6796.

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