Hitrap chelating hp columns

Protein purification was conducted after [19 (link)]. Briefly, overnight cultures of E. coli BL21-(DE3) pLysS were used to inoculate 1.0 litre overexpression cultures. These were then grown at 30°C to an OD₆₀₀ of 0.6, before protein expression was induced for 2 hours with 1mM IPTG. Cells were lysed using a cell disruptor (Avestin) and His₆-tagged proteins purified by NTA-Ni chromatography. 1 ml HiTrap chelating HP columns (Amersham) were equilibrated with 25 mM KH₂PO₄, 200 mM NaCl, pH 8.0 (SBW25 RimA/B/K), 50 mM Tris-Cl, 2.5% glycerol, pH 8.0 (SBW25 RpsF) or 50 mM Tris, 300 mM NaCl, 10 mM imidazole, pH 9.0 (E. coli RpsF), and loaded with cell lysate. Following protein immobilization, proteins were eluted with a linear gradient to 500 mM imidazole over a 15 ml elution volume.

1.0 litre E. coli BL21-(DE3) pLysS overexpression cultures were inoculated from overnights and grown at 30°C to an OD₆₀₀ of 0.6, before protein expression was induced for 2 hours with 1mM IPTG. Cells were then lysed by French press and His₆-tagged proteins purified by NTA-Ni chromatography. 1 ml HiTrap chelating HP columns (Amersham) were equilibrated with 25 mM KH₂PO₄, 200 mM NaCl, pH 8.0 (SBW25 RimA/B/K), 50 mM Tris-Cl, 2.5% glycerol, pH 8.0 (SBW25 RpsF) or 50 mM Tris, 300 mM NaCl, 10 mM imidazole, pH 9.0 (E. coli RimK and RpsF), and loaded with cell lysate. Following protein immobilization, elution was accomplished using a linear gradient to 500 mM imidazole over a 15 ml elution volume.

E. coli BL21-(DE3) pLysS overexpression cultures were inoculated from overnight cultures in a 1:100 ratio and grown at 37 °C to an OD₆₀₀ of 0.4, before protein expression was induced overnight with 0.5 mm isopropyl β-d-thiogalactopyranoside at 18 °C. Cells were then lysed by French press (3 × 20,000 p.s.i.) and centrifuged, and the proteins purified were from the supernatant by nickel-nitrilotriacetic acid chromatography. 1-ml HiTrap chelating HP columns (Amersham Biosciences) were equilibrated with 10 volumes of washing buffer (20 mm HEPES, pH 7.5, 250 mm NaCl, 2 mm MgCl₂, and 2.5% (v/v) glycerol, pH 7.5) and loaded with cell lysate. Following protein immobilization, the column was washed with 10 volumes of buffer containing 50 mm imidazole, before proteins were eluted using 500 mm imidazole buffer in a single step elution.