Hitraptm q hp column

The marine bacterium P. stutzeri 273 was cultured in glass flasks containing LB medium and incubated at 28°C under vigorous agitation for 48 h. Cell-free culture supernatant of the strain P. stutzeri 273 was collected by centrifugation at 12,000 rpm for 15 min, and precipitated overnight at 80% saturation with (NH₄)₂SO₄ at 4°C. The precipitate was collected by centrifugation and dissolved in 50 mM NaCl in 20 mM Tris-HCl (pH 9.0), and dialyzed against the same buffer overnight. The dialyzed fraction was loaded onto a 5 mL HiTrap^TM Q HP column (GE Healthcare) pre-equilibrated with 50 mM NaCl in 20 mM Tris-HCl (pH 9.0), then eluted with a NaCl gradient (50-500 mM) in the same buffer at 5 mL/min. Active fractions were collected, concentrated by ultra-filtration (100-kDa MW cut-off membrane, Millipore), and subjected to gel filtration on a Hiload^TM 16/600 Superdex^TM 200 column (GE Healthcare) pre-equilibrated with 150 mM NaCl in 20 mM Tris-HCl (pH 9.0). The column was eluted with the same buffer at a flow rate of 1 mL/min, and the active fractions were pooled for further analysis. All purification was performed at 4°C. Total sugar content was determined by the phenol-sulfuric acid method using glucose as the standard (Jiang et al., 2011 (link)), and active fractions were determined as described in the following section of inhibition assay of biofilm formation.

Amy63 was purified as described previously32 (link)33 (link) with minor modification. Briefly, marine bacterium strain 63 was propagated in marine broth 2216E containing 0.1% agar for 48 h at 28 °C. The culture broth was obtained by centrifugation at 8,000 rpm for 20 min at 4 °C and was adjusted to 80% saturation with ammonium sulfate. After precipitation overnight, the mixture was centrifuged (10,000 rpm g for 20 min) and the collected precipitate was dissolved in 10 mM Tris-HCl buffer (pH 8.0) and dialyzed against buffer A (100 mM NaCl in 10 mM Tris-HCl, pH 8.0) overnight. The dialyzed solution was applied to a HiTrap^TM Q HP column (GE Healthcare) pre-equilibrated with buffer A. Bound proteins were eluted with 150 ml linear gradient of 0.1–1.0 M of NaCl in 10 mM Tris-HCl (pH 8.0) at a flow rate of 5 ml/min. Active fractions were collected and concentrated by ultrafiltration (MWCO 10 kDa, Millipore), and loaded onto a Hiload^TM 16/600 superdex^TM 200 column (GE Healthcare) pre-equilibrated with 10 mM Tris-HCl (pH 8.0) containing 0.15 M NaCl. Bound proteins were eluted with the equivalent buffer at a flow rate of 1 ml/min (2 ml per tube). Active fractions were collected for further study. All purifications were carried out at 4 °C using an AKTA purifier system (Amersham Biosciences, Piscataway, NJ, USA).