Whole Cell Protein Extraction and Analysis

2.5 OD600 units of yeast cells were harvested by centrifugation for 5min with 3.000 g at room temperature. Cells were resuspended in 200μL 0.1M NaOH followed by incubation at room temperature for 5min. Whole cell extracts were pelleted for 5min with 3.000 g at room temperature and the pellet was resuspended in 50μL 1x SDS sample buffer. Whole cell extracts were separated by electrophoresis, transferred onto polyvinylidene difluoride membranes (Immobilon®-P PVDF Membrane, Sigma-Aldrich) and blocked in 5% skimmed milk dissolved in 0.05% Tween/PBS for 1h at room temperature. Membranes were incubated with primary antibodies overnight at 4°C followed by washing in 0.1% Tween/TBS. Membranes were incubated with appropriate HRP- linked secondary antibodies at 25°C for 1h and washed thrice prior to signal detection. Membranes were developed by chemiluminescence using ECL reagent.