DNA plasmids encoding the catalytic domain containing amino acid residues 111–340 of the original SB transposase sequence35 (link) or its hyperactive SB100X version33 (link) were ordered from GenScript USA (Piscataway, NJ). Plasmids were transformed into chemically competent BL21-A1 E. coli cells. Cells were grown in LB medium at 30oC in the presence of ampicillin at the concentration of 0.01 mg/mL until OD600 reached 0.6–0.8. Protein expression was induced by adding 1 mM IPTG and 0.2% L-arabinose for 4 hrs. Cells were collected by centrifugation and lysed in 50 mM TRIS and 500 mM NaCl lysis buffer at pH 8.0 by sonication. Soluble extract containing catalytic domain of SB transposase was prepared by centrifugation of cell lysate at 20,000 g for 1.5 hrs followed by overnight resuspension of pellet, and another centrifugation of cell lysate at 15,000g for 1hr. The protein was purified using Ni-NTA affinity resin (ClonTech, Mountain View, CA) and refolded by gradual decrease of urea concentration. The final sample was prepared in 25 mM TRIS buffer at pH 8.0. The presence and purity of proteins were monitored by SDS-page gel electrophoresis.
Ni nta affinity resin
Manufactured by Takara Bio
Sourced in United States
Ni-NTA affinity resin is a chromatographic medium used for the purification of recombinant proteins tagged with a histidine (His) sequence. It utilizes the high-affinity interaction between nickel ions (Ni2+) and the imidazole groups of the histidine residues to selectively bind and separate the target protein from other molecules in a sample.
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Lab products found in correlation
2 protocols using ni nta affinity resin
1
Purification of Hyperactive SB Transposase
2
Purification of Recombinant Proteins
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All manipulations were performed at 4°C. After cultivation, cells were collected by centrifugation at 13200 rpm for 5 min and washed twice with 0.9% NaCl. The pellets were resuspended in 0.5 ml of 0.1 M potassium phosphate (pH 7.5), 0.1 mM EDTA, and 0.4 mM dithiothreitol buffer solution with the addition of 0.1 mM phenylmethylsulfonyl fluoride. The obtained suspensions were sonicated and then centrifuged at 13200 rpm for 5 min. The supernatants were decanted and then subjected to enzymatic reactions and 12% SDS-PAGE. PageRuler Prestained Protein Ladder 26616 (Thermo Scientific) was used to evaluate protein molecular mass.
For protein purification, the cells were resuspended in 15 ml of the abovementioned buffer and disrupted with a French press. The cell debris was eliminated by centrifugation at 6000 rpm for 5 min. Then, the supernatant was applied to Ni-NTA Affinity Resin (Clontech, USA). The protein fractions were eluted with an imidazole gradient from 20 mM (in the binding buffer) to 500 mM (in the elution buffer). The fraction containing the purified protein was dissolved in a buffer with 50 mM Tris-HCl (pH 7.5), 500 mM NaCl and 5% glycerol.
For protein purification, the cells were resuspended in 15 ml of the abovementioned buffer and disrupted with a French press. The cell debris was eliminated by centrifugation at 6000 rpm for 5 min. Then, the supernatant was applied to Ni-NTA Affinity Resin (Clontech, USA). The protein fractions were eluted with an imidazole gradient from 20 mM (in the binding buffer) to 500 mM (in the elution buffer). The fraction containing the purified protein was dissolved in a buffer with 50 mM Tris-HCl (pH 7.5), 500 mM NaCl and 5% glycerol.
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