Sephadex g 25 nap columns

After exposure to the indicated treatments, samples from YPD liquid cultures were harvested, washed and resuspended at known cell densities (10–15 mg/ml, wet weight) in the extraction buffer, 100 mM 4-morpholine-ethanesulfonic acid (MES) pH 6.0, containing 5 mM cysteine and 0.1 mM phenyl methyl sulphonyl fluoride (PMSF). Cellular suspensions were transferred into small pre-cooled tubes (1.0 cm-diameter) with 1.5 g Ballotini glass beads (0.45 mm diameter). The cells were broken by vigorously vibrating the tubes in a vortex mixer and rapidly cooled in ice. The crude extract was then centrifuged at 10,000 ×g for 5 min and the pellet was resuspended in the same buffer at the initial density. For antioxidant assays, the supernatant fraction obtained was filtered through Sephadex G-25 NAP columns (Amersham Pharmacia Biotech AB) previously equilibrated with 50 mM K-phosphate buffer, pH 7.8, to remove low-molecular-weight compounds that could interfere with the measurements.

After exposure to different stresses, samples from the cultures were harvested and resuspended at known densities (10–15 mg/mL, wet weight) in the extraction buffer, 100 mM 4-morpholine-ethanesulfonic acid (MES) pH 6.0, containing 5 mM cysteine and 0.1 mM phenyl methyl sulphonyl fluoride (PMSF). Cellular suspensions were transferred into small pre-cooled tubes (1.0 cm diameter) with 1.5 g Ballotini glass beads (0.45 mm diameter). The cells were broken by vigorously vibrating the tubes in a vortex mixer. The tubes were rapidly cooled in ice. Then, the crude extract was centrifuged at 10,000× g for 5 min, and the pellet was resuspended in the same buffer at the initial density. For antioxidant assays, the supernatant fraction obtained was filtered through Sephadex G-25 NAP columns (Amersham Pharmacia Biotech AB, Staffanstorp, Sweden) previously equilibrated with 50 mM K-phosphate buffer, pH 7.8, to remove low-molecular-weight compounds.

After exposure to different stresses, samples from the cultures were harvested and resuspended at known densities (10–15 mg/mL wet weight) in the extraction buffer, 100-mM 4-morpholine-ethanesulfonic acid (MES) pH 6.0, containing 5-mM cysteine and 0.1-mM phenyl methyl sulphonyl fluoride (PMSF). The cellular suspensions were transferred into small precooled tubes (1.0-cm diameter) with 1.5-g Ballotini glass beads (0.45-mm diameter). Cells were broken by vigorously vibrating the tubes in a vortex mixer. The tubes were quickly cooled in ice. The crude extract was then centrifuged at 10,000× g for 5 min, and the pellet was resuspended in the same buffer at the initial density. For antioxidant assays, the supernatant fraction obtained was filtered through Sephadex G-25 NAP columns (Amersham Pharmacia Biotech AB) previously equilibrated with 50-mM K-phosphate buffer, pH 7.8, to remove low molecular weight compounds.