E. coli cells were lysed by sonication in 20 mM Tris buffer (pH 7.5) buffer A supplemented with protease inhibitor cocktail and DNase (Sigma, St. Louis, MO) and 200 mM NaCl. The cell lysate was clarified by centrifugation at 25,000 × g, and the supernatant was loaded onto a HisTrap HP column (GE healthcare, Marlborough, MA). The His6-tagged FRP was eluted with buffer A containing 300 mM imidazole and further purified by gel filtration chromatography by using a HiPrep Sephacryl S-200 HR (GE healthcare, Marlborough, MA) column and an isocratic flow of buffer A. The purity of FRP was confirmed by SDS–PAGE by using a precast gradient gel (Any kD™ Mini-PROTEAN, Bio-Rad, CA). The concentration of FRP was determined by 280 nm absorption on a NanoDrop spectrophotometer (Thermo Scientific, MA, USA).
Hiprep sephacryl s 200 hr
Manufactured by GE Healthcare
Sourced in United States
The HiPrep Sephacryl S-200 HR is a size exclusion chromatography column designed for the separation and purification of biomolecules such as proteins, peptides, and nucleic acids. It is capable of fractionating molecules within a broad molecular weight range.
Automatically generated - may contain errors
Lab products found in correlation
Nanodrop spectrophotometer, by Thermo Fisher Scientific (2 mentions)
Any kd mini protean, by Bio-Rad (1 mentions)
Histrap hp column, by GE Healthcare (1 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
Frac30 fraction collector, by GE Healthcare (1 mentions)
Hitrap heparin hp, by GE Healthcare (1 mentions)
3 protocols using hiprep sephacryl s 200 hr
1
Purification of Flavin Reductase Protein
2
ÄKTA Start Protein Purification System
3
Purification and Tag Removal of pyCyp Protein
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The supernatant from the previous step was mixed with Ni–NTA affinity resin. The mixture was stirred for 1 h at 4 °C and loaded into the column. An unbound protein was eluted with lysis buffer containing 20 mM of imidazole, and pyCyp was eluted with lysis buffer containing 250 mM of imidazole. To remove the tag of hexa-histidine, the eluate was treated with TEV protease and incubated overnight at room temperature. The pyCyp protein were concentrated using Centriprep (GE Healthcare, Chicago, IL, USA), and purified using HiPrep Sephacryl S-200 HR (GE Healthcare, Chicago, IL, USA) pre-equilibrated with lysis buffer. Purified pyCyp was concentrated to 10 mg/mL in 20 mM of Tris buffer (pH 8.0, 150 mM of NaCl and 2 mM of 2-mercaptoethnol). It was stored frozen at 4 °C until it was used in the experiment, and the concentration was measured by the Bradford assay. The yield was around 10 mg/L of culture medium.
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