1 ml resource rpc column

V. fluvialis LMGT 4216 was cultivated in 1 L of BHI broth at 30°C for 24 h. Cells were removed by centrifugation (10,000 × g for 30 min at 4°C), and the bacteriocin was precipitated from the culture supernatant with ammonium sulfate (60% saturation at 4°C overnight). The precipitate was harvested by centrifugation (15,000 × g for 40 min at 4°C), redissolved in 700 mL of distilled water, and adjusted to pH 3.5 with 1 M hydrochloric acid. The sample was applied to a Hi-Prep 16/10 SP-XL column (GE Healthcare, Chicago, IL, USA). Unbound material was washed from the column with 150 mL of 25 mM sodium citrate-phosphate buffer (pH 3.5). The bacteriocin was eluted with 100 mL of 0.5 M sodium chloride, and the eluate was then applied to a 1-mL Resource RPC column (GE Healthcare) connected to an Äkta purifier system (Amersham Pharmacia Biotech, Amersham, UK). The column was previously equilibrated with 0.1% (vol/vol) trifluoroacetic acid (TFA), and the bacteriocin was eluted from the column using a linear gradient (40 column volumes [CV]) of isopropanol containing 0.1% (vol/vol) TFA at 1 mL/min.

V. fluvialis LMGT 4216 was cultivated in 1 L of BHI broth at 30°C for 24 h. Cells were removed by centrifugation (10,000 × g for 30 min at 4°C), and the bacteriocin was precipitated from the culture supernatant with ammonium sulfate (60% saturation at 4°C overnight). The precipitate was harvested by centrifugation (15,000 × g for 40 min at 4°C), redissolved in 700 mL of distilled water, and adjusted to pH 3.5 with 1 M hydrochloric acid. The sample was applied to a Hi-Prep 16/10 SP-XL column (GE Healthcare, Chicago, IL, USA). Unbound material was washed from the column with 150 mL of 25 mM sodium citrate-phosphate buffer (pH 3.5). The bacteriocin was eluted with 100 mL of 0.5 M sodium chloride, and the eluate was then applied to a 1-mL Resource RPC column (GE Healthcare) connected to an Äkta purifier system (Amersham Pharmacia Biotech, Amersham, UK). The column was previously equilibrated with 0.1% (vol/vol) trifluoroacetic acid (TFA), and the bacteriocin was eluted from the column using a linear gradient (40 column volumes [CV]) of isopropanol containing 0.1% (vol/vol) TFA at 1 mL/min.