Ni nta agarose

Manufactured by Bio-Rad

Sourced in United States, Japan, China

Ni-NTA agarose is an affinity chromatography resin used for the purification of recombinant proteins with a histidine-tag. It consists of nickel-charged nitrilotriacetic acid (Ni-NTA) immobilized on cross-linked agarose beads. The histidine-tag binds to the Ni-NTA, allowing the target protein to be separated from other cellular components during the purification process.

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Lab products found in correlation

Ni nta agarose, by Qiagen (4 mentions) Poly prep chromatography column, by Bio-Rad (3 mentions) Ni nta slurry, by Qiagen (1 mentions) 0.45 μm filter, by Merck Group (1 mentions) Millex syringe filter unit, by Merck Group (1 mentions) Glutathione agarose 4b, by Macherey-Nagel (1 mentions) Amicon ultra10 filters, by Merck Group (1 mentions) Anti flag, by Beyotime (1 mentions) Polyprep columns, by Bio-Rad (1 mentions) Sypro ruby protein gel stain, by Thermo Fisher Scientific (1 mentions) Hispur cobalt purification kit, by Thermo Fisher Scientific (1 mentions) Bca protein assay kit, by Beyotime (1 mentions) Econo column, by Bio-Rad (1 mentions)

10 protocols using ni nta agarose

Cloning and Purification of relA and relQ

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The coding sequences of relA and relQ were amplified from the genomic DNA of S. suis SC-19 using primers relAF/relAR and relQF/relQR (see Supplementary Table S7), respectively. The primers were designed according to the sequences of genes SSU05_2094 and SSU05_1060 of S. suis 05ZYH33 (GenBank accession no. CP000407), and cloned into a prokaryotic expression vector pET-28a (Novagen, Shanghai, China), respectively. The resultant plasmids pET28a-relA and pET28a-relQ were confirmed by DNA sequencing and transformed into E. coli BL21(DE3) for expression of His-tagged recombinant proteins, respectively. The bacteria were induced by 1 mM isopropyl-beta-D-thiogalactopyranoside (IPTG) at 37 °C for 3 h. Purification of the recombinant proteins was achieved using Ni-NTA agarose (Bio-Rad, Shanghai, China) under native condition according to the manufacturer’s instructions. Electrophoresis was carried out with 12% SDS-PAGE.

Zhang T., Zhu J., Wei S., Luo Q., Li L., Li S., Tucker A., Shao H, & Zhou R. (2016). The roles of RelA/(p)ppGpp in glucose-starvation induced adaptive response in the zoonotic Streptococcus suis. Scientific Reports, 6, 27169.

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Purification of E. coli RNAP

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A strain of Eco containing plasmid with subunits of the RNAP core (LK1853¹) was grown to the exponential phase (OD₆₀₀ ~ 0.5). Expression of RNAP was induced with 500 μM IPTG for 4 h at room temperature. Cells were harvested by centrifugation, washed, resuspended in P buffer (300 mM NaCl, 50 mM Na₂HPO₄, 5% glycerol, 3 mM β-mercaptoethanol), and disrupted by sonication. Cell debris was removed by centrifugation and supernatant was mixed with 1 ml Ni-NTA Agarose (Qiagen) and incubated for 90 min at 4 °C with gentle shaking. Ni-NTA Agarose with bound RNAP was loaded on a Poly-Prep® Chromatography Column (BIO-RAD), washed with P buffer and, subsequently, washed with P buffer with 30 mM imidazole. The proteins were eluted with P buffer containing 400 mM imidazole and fractions containing RNAP were pooled and dialyzed against storage buffer (50 mM Tris–HCl, pH 8.0, 100 mM NaCl, 50% glycerol, 3 mM β-mercaptoethanol). The RNAP protein was stored at −20 °C.

Kouba T., Koval’ T., Sudzinová P., Pospíšil J., Brezovská B., Hnilicová J., Šanderová H., Janoušková M., Šiková M., Halada P., Sýkora M., Barvík I., Nováček J., Trundová M., Dušková J., Skálová T., Chon U., Murakami K.S., Dohnálek J, & Krásný L. (2020). Mycobacterial HelD is a nucleic acids-clearing factor for RNA polymerase. Nature Communications, 11, 6419.

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Purification of β-Lactamase from E. coli

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E. coli DH5α cells carrying the pSEM3132 plasmid were grown overnight in 150 ml LB containing 100 μg/ml ampicillin. Cells were removed by centrifugation and the cleared supernatant was filtered using a 0.45 μm filter (Millipore). 2 ml of Ni-NTA slurry (Qiagen) was added to the solution, followed by 1 h incubation at 4 °C. A Poly-Prep Chromatography Column (BIO-RAD) was used to collect the Ni-NTA agarose-bound proteins from the mixture. Twenty column volume of washing buffer (50 mM sodium phosphate, pH 7.2, 600 mM NaCl, 10% glycerol) was allowed to flow through the column. β-lactamase was eluted by four column volumes of elution buffer (50 mM sodium phosphate, pH 7.2, 600 mM NaCl, 50% glycerol) containing 250 mM imidazole. The solution was filtered using a 0.2 μm filter (Millipore) and stored at −20 °C. 10 μl of the solution was plated on an LB agar plate containing 100 μg/ml ampicillin, and no colony growth was observed after 10 days of incubation at 37 °C.

Svenningsen M.S., Veress A., Harms A., Mitarai N, & Semsey S. (2019). Birth and Resuscitation of (p)ppGpp Induced Antibiotic Tolerant Persister Cells. Scientific Reports, 9, 6056.

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Purification of Cas1 Protein Variants

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Strains E. coli BL21 (DE3) pET30a::cas1, E. coli BL21 (DE3) pET30a::cas1^E149A, E. coli BL21 (DE3) pET30a::cas1^H206A, and E. coli BL21 (DE3) pET30a::cas1^E221A were grown overnight in LB medium containing 50 μg/ml Kan. LB broth containing Kan was inoculated to an OD₆₀₀ of 0.05 with the overnight culture, and the cells were grown at 37°C with shaking until the culture density reached an OD₆₀₀ of 0.5. Expression was induced with 0.4 mM (final concentration) IPTG, and the cells were grown for an additional 6 h at 37°C. The induced cultures were harvested, and the cells were resuspended in 50 ml of buffer (pH 8.0, 300 mM NaCl, 50 mM NaH₂PO₄, 5 mM imidazole) and sonicated. The supernatant of cell lysates was collected by centrifugation and filtered through a 0.45-μm Millex syringe filter unit (Millipore, USA). The filtrate was subjected to nickel-chelate affinity chromatography (Ni-NTA agarose, Bio-Rad, USA) by following the manufacturer's instructions. Elution of the target protein was performed by competition with increasing concentrations of imidazole. Products from the course of purification were collected and measured by SDS-PAGE, and the protein concentration was determined using the Pierce BSA Protein Assay Kit (Pierce Biotechnology, USA). The purified target protein was dialyzed in 50 mM NaH₂PO₄ buffer (pH 8.0).

He Y., Wang M., Liu M., Huang L., Liu C., Zhang X., Yi H., Cheng A., Zhu D., Yang Q., Wu Y., Zhao X., Chen S., Jia R., Zhang S., Liu Y., Yu Y, & Zhang L. (2018). Cas1 and Cas2 From the Type II-C CRISPR-Cas System of Riemerella anatipestifer Are Required for Spacer Acquisition. Frontiers in Cellular and Infection Microbiology, 8, 195.

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Purification of Recombinant Proteins in E. coli

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The recombinant protein GST-NC2β, GST-GFP and His-ARP8 were expressed in E. coli BL21 cells, and the soluble recombinant proteins were purified as described previously (50 (link)). Briefly, the transformed bacteria were resuspended in lysis buffer containing 20 mM Tris–HCl (pH 7.3), 500 mM NaCl, 10% glycerol, 0.1% Triton X-100 and 1 mM PMSF, then disrupted by ultrasonication. The supernatant was collected and recombinant proteins were purified using an Ni-NTA agarose (Bio-Rad) or glutathione agarose 4B (Macherey-Nagel) affinity column. After washing with lysis buffer containing 0.3% GSH (m/v) or an increasing concentration gradient of imidazole (150, 200, 250, 300 and 400 mM), proteins were eluted and concentrated with Amicon-Ultra-10 filters (Millipore).

Cui G., Dong Q., Duan J., Zhang C., Liu X, & He Q. (2020). NC2 complex is a key factor for the activation of catalase-3 transcription by regulating H2A.Z deposition. Nucleic Acids Research, 48(15), 8332-8348.

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Purification and Detection of Cas1-Cas2 Complex

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The Cas1-His and Cas2-FLAG proteins were expressed from pET30a::cas1-cas2 in E. coli BL21 (DE3). Cell lysates of induced cultures (~10⁸ CFU) were incubated with 50 μl of Ni-NTA agarose (Bio-Rad) that was pre-equilibrated with binding buffer (50 mM NaH₂PO₄, 300 mM NaCl, pH 8.0) for 16 h at 4°C. After incubation, the resin was washed four times with binding buffer containing 10 mM imidazole to remove non-specifically bound proteins. The resin was then resuspended in 30 μl of SDS loading buffer, heated to 100°C for 5 min and used for Western blot analysis using the anti-FLAG (Beyotime, China) as a primary antibody.

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Purification of Recombinant Plant Aquaporins

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The solubilized proteins were mixed with 10 mM imidazole and 4 mL of Ni-NTA agarose (Qiagen, Hilden, Germany) preequilibrated with buffer A + 3 × CMC OG and incubated overnight at 4 °C. Ni-NTA agarose with proteins was packed into empty PolyPrep-columns (Bio-Rad, Tokyo, Japan) and washed with 10-bed volumes of buffer B [20 mM HEPES-NaOH pH 7.8, 300 mM NaCl, 10% (v/v) glycerol, 5 mM β-mercaptoethanol] with 3 × CMC OG and 30 mM imidazole. The proteins were eluted in buffer B with 3 × CMC OG and 300 mM imidazole in the first elution and 500 mM in the second. Fractions were analyzed by Coomassie and Western-Blot, and protein concentration was determined by A₂₈₀ in Nanodrop (extinction coefficient of 46.41 M⁻¹cm⁻¹ for BoPIP1;2 and 46.87 M⁻¹cm⁻¹ for BoPIP2;2, and molecular weights of 33.73 kDa and 33.14 kDa) [36 ].

Yepes-Molina L., Teruel J.A., Johanson U, & Carvajal M. (2024). Brassica oleracea L. var. italica Aquaporin Reconstituted Proteoliposomes as Nanosystems for Resveratrol Encapsulation. International Journal of Molecular Sciences, 25(4), 1987.

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Purification of Recombinant HSulf-1 Protein

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Culture media from 293T cells transiently transfected with pcDNA3.1/Myc-His(−)-HSulf-1 plasmid was incubated with Ni-NTA agarose (Qiagen, Venlo, Netherlands) in the presence of 15 mM imidazole at 4°C overnight. The mixture was poured into a poly-prep chromatography column (Bio-Rad, Hercules, CA) and the media were flowed through the column to capture Ni-NTA agarose bound rHSulf1. The beads were washed with three times of column volume of washing buffer (50 mM NaH₂PO₄, 300 mM NaCl, 20 mM imidazole, 0.05% Tween 20, pH 8) and the bound rHSulf1 was eluted with five times of column volume of elution buffer (50 mM NaH₂PO₄, 300 mM NaCl, 250 mM imidazole, 0.05% Tween 20, pH 8). The eluted rHSulf1 was concentrated using Amicon Ultra-0.5 ml, 50 MWCO centrifuge filter units (EMD Millipore, Darmstadt, Germany), washed into phosphate-buffered saline and purified again using HisPur Cobalt purification kit (Thermo Fisher Scientific Inc., Waltham, MA) according to manufacturer instruction. The final eluted fraction was analyzed using gel electrophoresis and subjected to SYPRO^® Ruby Protein Gel Stain (Thermo Fisher Scientific Inc., Waltham, MA). The protein was quantified by comparing the band intensity with a BSA standard curve using ImageJ (National Institutes of Health, Bethesda, MD) [23 (link)].

Yang Y.W., Phillips J.J., Jablons D.M, & Lemjabbar-Alaoui H. (2020). DEVELOPMENT OF NOVEL MONOCLONAL ANTIBODIES AND IMMUNOASSAYS FOR SENSITIVE AND SPECIFIC DETECTION OF SULF1 ENDOSULFATASE. Biochimica et biophysica acta. General subjects, 1865(2), 129802.

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Purification of Recombinant Proteins

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The plasmids pET30a-mhp390 and pET30a-NOD1 (EcoRI and XhoI restriction sites, primers listed in Table S1) were transformed into E. coli BL21 (DE3) to express the recombinant protein. The resultant recombinant protein tagged with 6×His was purified with Ni-NTA agarose (Bio-Rad, Shanghai, China). The concentration of the purified protein was measured by using the BCA Protein Assay Kit (Beyotime, China).

Liu W., Jiang P., Yang K., Song Q., Yuan F., Liu Z., Gao T., Zhou D., Guo R., Li C., Sun P, & Tian Y. (2022). Mycoplasma hyopneumoniae Infection Activates the NOD1 Signaling Pathway to Modulate Inflammation. Frontiers in Cellular and Infection Microbiology, 12, 927840.

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Purification of Recombinant Protein CK-2

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Fifty milliliters of overnight culture grown at 37 o C was used to inoculate 500 ml of LB broth. The cells were grown until the OD600 reached 0.5 and then induced with Isopropyl β-D-1-thiogalactopyranoside (IPTG) at a final concentration of 1 mM. Following induction, the cells were grown for another 5 h and harvested by centrifugation at 10,000 rpm for 10 min. Protein was purified using Ni-NTA Agarose (Qiagen, Mississauga, ON) according to manufacturer's instruction. Briefly, one ml of Ni-NTA Agarose resin was added to 10 ml of cleared cell lysate and incubated for 1 h at 4 C. After incubation, the lysate was loaded onto a 25 ml econocolumn (Bio-Rad) and washed with binding buffer (100 mM NaH2PO4, 10 mM Tris, 8 M urea) equilibrated to pH 8.0, pH 6.3, and pH 5.9. Finally, recombinant CK-2 (rCK-2) was eluted at pH 4.5 and was stored at 4 o C. The elution fractions were separated on a 17% sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis. Following SDS-PAGE, the proteins were transferred onto a nitrocellulose membrane blocked with 5% skim milk in TBS-T buffer (0.14 M NaCl, 2.7 mM KCL, 25 mM Tris, 0.5% Tween 20, pH 8) for 1 h. The membrane was washed with TBS-T for 5 min and probed with 1:5000 dilution of mouse anti-
express antiserum (Invitrogen, Carlsbad, CA) to identify the specific Xpress epitope tag on the recombinant protein.

Semple S.L., Eshaque S., Fujiki K., Tang C., Mitchell L., Bols N.C, & Dixon B. (2018). CK-2 of rainbow trout (Oncorhynchus mykiss) has two differentially regulated alleles that encode a functional chemokine. Veterinary immunology and immunopathology, 198.

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