Tris hcl ph 6.8

After yeast was cultured in syringes, the appropriate number of cells was collected and they were suspended in 100 µL of 0.2 m NaOH (Wako) and 1% 2‐mercaptoethanol (Wako) per OD, placed on ice for 10 min, and centrifuged at 17 800 g at 4 °C for 2 min. The supernatant was discarded, and 100 µL of 1× sample buffer (2% SDS (Nacalai Tesque), 100 mm DTT (Wako), 60 mm Tris/HCl (pH 6.8) (Sigma), 0.001% bromophenol blue (Sigma), 10% glycerol (Wako)) was added per 1 OD. The pellet was suspended by adding 100 µL per OD, heated at 100 °C for 5 min, and the centrifuged supernatant was used as the sample for SDS/PAGE.

Standard procedures were applied for the Western blot assay [21 (link)]. Cells were harvested by cell dissociation buffer and collected by centrifugation. Harvested cells were lysed in 0.125 M Tris–HCl pH 6.8 (Sigma–Aldrich), 5% sodium dodecyl sulfate (SDS, Lonza, Verviers, Belgium), and protease/phosphatase inhibitors (25 mM sodium fluoride, 10 μg/mL pepstatin A, 1 mM phenylmethylsulfonyl fluoride, 10 μg/mL trypsin inhibitor, 12.5 μg/mL leupeptin, 30 μg/mL aprotinin, 1 mM sodium orthovanadate, and 1 mM sodium molibdate, all purchased from Sigma–Aldrich). Following sonication and boiling, protein content was determined through the bicinchoninic acid (BCA) assay method (Pierce, Thermo Fisher Scientific, Waltham, MA), and SDS-PAGE was performed. Proteins were then transferred onto nitrocellulose membranes. The films were incubated in “Blocking buffer” (PBS-Tween containing 5% (w/v) dried skimmed milk) for 1 h and incubated overnight at 4 °C with the primary antibodies dissolved in the blocking buffer. Following 1 h incubation at room temperature with horseradish-conjugated secondary antibodies, membranes were exposed to chemiluminescence solution (ECL, GE Healthcare).