Ni2 nitrilotriacetic acid agarose

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Ni2+-nitrilotriacetic acid-agarose is a chromatography resin used for the purification of proteins. It consists of nickel-nitrilotriacetic acid immobilized on an agarose matrix. This resin binds to proteins with a specific affinity tag, such as a histidine tag, allowing for selective capture and purification of the target protein.

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Ni2+-nitrilotriacetic acid (Ni-NTA)-agarose resin Ni2+-nitrilotriacetic acid agarose chelation chromatography Ni2+-nitrilotriacetic acid agarose beads Ni2+-NTA (nitrilotriacetic acid) agarose resin Ni2+-nitrilotriacetic acid (NTA) agarose Agarose

6 protocols using ni2 nitrilotriacetic acid agarose

Purification of Recombinant His-Tagged PTB Protein

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Recombinant His-PTB was purified as described earlier (9). Briefly, recombinant PTB was transformed in Escherichia coli (BL21 DE3) cells and the expression was induced by 0.6 mM IPTG. For purifying the protein, the lysate was incubated with Ni²⁺–nitrilotriacetic acid–Agarose (Qiagen) under non-denaturing conditions at 4°C and eluted with 250 mM imidazole (9 (link)).

Katoch A., George B., Iyyappan A., Khan D, & Das S. (2017). Interplay between PTB and miR-1285 at the p53 3′UTR modulates the levels of p53 and its isoform Δ40p53α. Nucleic Acids Research, 45(17), 10206-10217.

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Purification and Preservation of NhaA

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Overexpression of NhaA^{33 (link)}, isolation of high-pressure membranes^{39 (link)}, and affinity purification (Ni²⁺-nitrilotriacetic acid-agarose, Qiagen)^{40 (link)} were performed as described previously, but the protein was eluted in a buffer (pH 7.5) containing 300 mM imidazole, 25 mM citric acid, 100 mM choline chloride, 5 mM MgCl₂, and 0.015% n-dodecyl β-D-maltopyranoside (DDM). Sucrose (10%) was added to the eluted protein solution, and the solution was dialyzed overnight at 4 °C in acidic dialysis buffer containing 10% sucrose, 100 mM choline chloride, 25 mM citric acid, 5 mM MgCl₂, 0.015% DDM (pH 4.0), and was frozen at −80 °C.

Rimon A., Mondal R., Friedler A, & Padan E. (2019). Cardiolipin is an Optimal Phospholipid for the Assembly, Stability, and Proper Functionality of the Dimeric Form of NhaA Na+/H+ Antiporter. Scientific Reports, 9, 17662.

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Purification of NhaA Membrane Protein

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Overexpression of NhaA^{58 (link)}, isolation of high-pressure membrane vesicles^{57 (link)}, and immobilized metal-affinity purification (Ni²⁺-nitrilotriacetic acid-agarose, Qiagen)^{59 (link)} were performed as described previously, but the protein was eluted in a buffer (pH 7.9) containing 10% glycerol, 300 mM imidazole, 25 mM citric acid, 100 mM choline chloride, 5 mM MgCl₂, and 0.015% DDM. Sucrose (10%) was added to the eluted protein solution, and the solution was dialyzed overnight at 4 °C in acidic dialysis buffer containing 25 mM potassium citrate (pH 4.0), 10% sucrose, 100 mM choline chloride, 25 mM citric acid, 5 mM MgCl₂, 0.015% DDM and was frozen at – 80 °C.

Quick M., Dwivedi M, & Padan E. (2021). Insight into the direct interaction of Na+ with NhaA and mechanistic implications. Scientific Reports, 11, 7045.

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Purification of Recombinant Human Ribosomal Protein S5

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Human ribosomal protein S5 was expressed in BL21 DE3 and purified as mentioned earlier (17 (link)). In brief, Escherichia coli BL21 DE3 cells were transformed with the plasmid pET-28(a)-WTRPS5 or pET-28(a)RPS5-βhpt expressing the N terminally poly(His)‐tagged wild type or mutant human RPS5 protein. Protein expression was induced by 0.4 mM Isopropyl β-D-1-thiogalactopyranoside (IPTG) and incubated at 16°C for 22 h. Later protein was purified using Ni²⁺–nitrilotriacetic acid–agarose (Qiagen, Hilden, Germany) under non‐denaturing conditions and eluted with elution buffer containing 250 and 500 mM imidazole.

Bhat P., Shwetha S., Sharma D.K., Joseph A.P., Srinivasan N, & Das S. (2015). The beta hairpin structure within ribosomal protein S5 mediates interplay between domains II and IV and regulates HCV IRES function. Nucleic Acids Research, 43(5), 2888-2901.

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Purification of Recombinant PTB and JEV NS5

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E. coli BL21 (DE3) cells were transformed with the expression vector pET28a-PTB (a generous gift from Dr. J.G. Patton) and pET28a-NS5 (a generous gift from Dr. R. Chang), and the expression of his-tagged recombinant PTB or JEV NS5, respectively, was induced by 1 mM IPTG. The recombinant protein was purified using Ni²⁺-nitrilotriacetic acid-agarose (Qiagen) under non-denaturing conditions and eluted with 250 mM imidazole. The protein was electrophoresed on a 10% Sodium dodecyl sulphate (SDS)-poly acrylamide gel (PAGE) to confirm the size and the purity. Protein concentration was determined by the BCA method and the aliquots were snap frozen and stored at −70°C. Freshly-thawed protein aliquot was used for all assays.

Bhullar D., Jalodia R., Kalia M, & Vrati S. (2014). Cytoplasmic Translocation of Polypyrimidine Tract-Binding Protein and Its Binding to Viral RNA during Japanese Encephalitis Virus Infection Inhibits Virus Replication. PLoS ONE, 9(12), e114931.

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Purification of Recombinant GAPDH Proteins

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BL21 (DE3) pLysS cells (EMD4 Millipore, Billerica, MA) that contained the recombinant plasmid pET100/d-TOPO GAPDH/GAPDH (Pro²³⁴Ser) or pGEX-2T GAPDH/GAPDH (Pro²³⁴Ser) were grown at 37 °C to 0.6_A600, and then induced with 0.1 mM isopropyl β-D-thiogalactopyranoside for 4 h at 32 °C. The liquid culture was centrifuged at 4300g for 30 min and the pellet resuspended in cold PBS, 0.1% Triton X-100, 1 mM PMSF, sonicated, centrifuged at 17,211g for 20 min, and then the supernatant applied to a 1 ml column of Ni²⁺-nitrilotriacetic acid-agarose (Qiagen, Valencia, CA) equilibrated in Buffer A (10 mM Hepes (pH 7.9), 5 mM MgCl₂, 0.1 mM EDTA, 50 mM NaCl, and 0.8 mM imidazole). The column was washed with 10 volumes of Buffer A containing 25 mM imidazole. The tagged protein was eluted with Buffer A supplemented with 200 mM imidazole. Alternatively, the lysate was applied to a PBS washed glutathione-Sepharose 4B column and eluted with 5 mM reduced glutathione [29 ]. The eluted recombinant proteins were dialyzed against the appropriate buffer, concentrated using a Centriprep centrifugal filter device (EMD Millipore), and then the total protein concentration determined by BCA protein assay (Pierce, Rockford, IL).

Tisdale E.J., Talati N.K., Artalejo C.R, & Shisheva A. (2016). GAPDH binds Akt to facilitate cargo transport in the early secretory pathway. Experimental cell research, 349(2), 310-319.

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