N-terminally 6xHis-tagged E. coli RF1 was overexpressed in BL21 E. coli cells grown at 37°C from overnight culture in LB medium and in presence of 100 μg/mL Ampicillin. Protein expression was induced at A600 of 0.4 by adding IPTG to a final concentration of 1 mM. RF1 was expressed from pET28-plasmid kindly provided by Rachel Green (John Hopkins University, Baltimore). After 1 h of expression, cells were lysed using a microfluidizer. The cell lysate was cleared by centrifugation in a SS34 rotor (Sorval) at 4°C and 44,100 x g for 30 min. Purification of His-tagged RF1 was done with Protino Ni-NTA agarose beads (Macherey-Nagel). The final eluate was applied onto a Superdex HiLoad S75 16/600 column (GE Healthcare) to yield the final concentrated protein in gel filtration buffer (50 mM HEPES pH 7.4, 50 mM KCl, 100 mM NaCl, 2% glycerol and 5 mM 2-mercaptoethanol).
Protino ni nta agarose beads
Manufactured by Macherey-Nagel
Sourced in Germany
Protino Ni-NTA agarose beads are affinity chromatography media designed for the purification of His-tagged recombinant proteins. The beads are composed of agarose matrix with chelated nickel ions, which bind to the histidine residues of the target protein. This allows for selective capture and efficient purification of His-tagged proteins from complex samples.
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Lab products found in correlation
Superdex hiload s75 16 600 column, by GE Healthcare (2 mentions)
Pierce universal nuclease, by Thermo Fisher Scientific (1 mentions)
Micro bca protein assay kit, by Thermo Fisher Scientific (1 mentions)
Pierce protein concentrator, by Thermo Fisher Scientific (1 mentions)
Chaperone plasmid set, by Takara Bio (1 mentions)
Horseradish peroxidase conjugated goat anti rabbit igg antibody, by Merck Group (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Agarose, by Merck Group (1 mentions)
Imidazole, by Merck Group (1 mentions)
Cnbr activated sepharose beads, by GE Healthcare (1 mentions)
Purified human igg, by Merck Group (1 mentions)
Edta free protease inhibitor cocktail, by Roche (1 mentions)
5 protocols using protino ni nta agarose beads
1
Purification of Recombinant E. coli RF1
2
Recombinant Protein Expression and Purification
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Full-length coding sequences were amplified using Primers 8 and 9 for DLK2 and Primers 10 and 11 for D14 from Ler cDNA (Supplementary Table S1 ), and were ligated into the NdeI and BamHI or NdeI and EcoRI sites of pET-28c vector (Novagen, United States). Clones were sequenced and transformed into Rosetta DE3 pLysS cells (Novagen, United States). Protein expression was induced with 1 mM IPTG when the optical density at 600 nm reached 0.8, and incubated overnight (16 h) at 18°C/200 rpm. Harvested cultures were washed with NPI-10 buffer (50 mM NaH2PO4, 300 mM NaCl, 10 mM imidazole, pH 8.0) and stored at -80°C. Pellets were resuspended in NPI-10 buffer supplemented with 1 mg/mL lysozyme and 3 units/mL Pierce Universal Nuclease (Thermo Fischer Scientific, United States). Clarified lysates were batch purified using Protino Ni-NTA agarose beads (Macherey-Nagel, Germany). Bound proteins were eluted with NPI-250 buffer (50 mM NaH2PO4, 300 mM NaCl, 250 mM imidazole, pH 8.0), buffer-exchanged into 20 mM HEPES, pH 7.5, 150 mM NaCl, and 10% (v/v) glycerol and concentrated using Pierce Protein Concentrator (10 kDa; Thermo Fischer Scientific, United States). Protein concentration was estimated with Micro BCA Protein Assay Kit (Thermo Fischer Scientific, United States) and adjusted to 2 mg/mL. Protein purity was assessed by SDS-PAGE (Supplementary Figure S1 ).
3
Purification of Recombinant E. coli RF1
4
Purification of His-tagged Proteins
5
Membrane Protein Extraction and Purification
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The envelope fraction was resuspended at a concentration of approximately 10 mg/ml in solubilization buffer ( To facilitate extraction of membrane proteins, samples were subjected to mild agitation for 1 hour at 4°C. Insoluble material was removed by centrifugation at 16000x g at 4°C. To perform IgG affinity purification, membrane-extracted proteins were incubated for 1.5 hours at 4°C with purified human IgG (Sigma) that had been previously coupled to CNBr-activated Sepharose beads (GE Healthcare). After extensive washes of the resin with solubilization buffer containing 0.3% (w/v) digitonin and 0.03% (w/v) DDM, bound proteins were eluted by incubation with
AcTEV protease (ThermoFisher) overnight at 4°C under mild agitation. To perform nickel (Ni)-affinity purification, membrane-extracted proteins were supplemented with 20 mM imidazole and incubated with Protino Ni-NTA agarose beads (Macherey-Nagel) for 1 hour at 4°C. After extensive washes of the resin with solubilization buffer supplemented with 50 mM imidazole, 0.3% (w/v) digitonin, 0.03% (w/v) DDM, and the EDTA-free protease inhibitor cocktail (Roche), bound proteins were eluted using the same buffer supplemented with 500 mM imidazole.
AcTEV protease (ThermoFisher) overnight at 4°C under mild agitation. To perform nickel (Ni)-affinity purification, membrane-extracted proteins were supplemented with 20 mM imidazole and incubated with Protino Ni-NTA agarose beads (Macherey-Nagel) for 1 hour at 4°C. After extensive washes of the resin with solubilization buffer supplemented with 50 mM imidazole, 0.3% (w/v) digitonin, 0.03% (w/v) DDM, and the EDTA-free protease inhibitor cocktail (Roche), bound proteins were eluted using the same buffer supplemented with 500 mM imidazole.
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