Chemostar touch ecl fluorescence imager

About 50 ml yeast cultures were grown to an absorbance of 1.0 ± 0.2 at 600 nm, harvested (4000g, 5 min), washed with 20 ml of ice-cold water, and resuspended in 500 μl buffer (5 mM EDTA, 25 mM Tris, and pH 7.5). The cells were disrupted by vortexing with 500 μg acid-washed glass beads (450–600 μm; Sigma–Aldrich) for 30 s and cooling on ice for 1 min with 12 repetitions. The supernatant was collected (4000g, 5 min), and the procedure was repeated with the remaining pellet. The microsomal fraction was obtained by removal of high-density components (10,000g, 5 min, 4 °C) and ultracentrifugation (100,000g, 45 min, 4 °C). The membrane pellets were resuspended in 0.1 ml of 50 mM NaCl, 100 mM sodium phosphate, pH 8.0. About 7.5 μg of total protein (Bradford Protein Assay; Bio-Rad) were separated by SDS-PAGE and blotted (semidry; Bio-Rad) on Polyvinylidene fluoride membranes (Hybond P 0.45; GE Healthcare). A monoclonal mouse antihemagglutinin antibody (catalog number: 34660; Qiagen) and a horseradish peroxidase–conjugated secondary goat–antimouse antibody (catalog number: 115-035-174; Jackson ImmunoResearch) were used for detection with the Clarity ECL system (Bio-Rad). Signals were monitored using a Chemostar Touch ECL & Fluorescence Imager (Intas Science Imaging Instruments).