Whole-Cell Yeast Protein Extraction and Western Blot

Whole-cell yeast extracts were prepared using the trichloroacetic acid method as previously described (Chen et al., 2012 (link)). The pelleted cells from 5 ml of culture were washed once with water and resuspended in 10% trichloroacetic acid. The cells were lysed by vortexing with glass beads, and the protein lysates were pelleted by centrifugation at 12,000 g for 15 min. The pellets were washed with ice-cold 80% acetone, and proteins were dissolved in ×2 SDS sample loading buffer by boiling for 5 min. The samples were centrifuged for 5 min at 12,000 g, and the supernatant was retained as protein extract. The samples were resolved on 8% SDS-PAGE gel and transferred onto a polyvinylidene difluoride membrane (Immobilon-P; Millipore) using a semi-dry method. Anti-Myc and anti-FLAG antibodies were purchased from MBL. GAPDH was purchased from GeneTex. Anti-mouse and rabbit IgG HRP-conjugated secondary antibodies were purchased from Santa Cruz Biotechnology. Blots were developed using the Western Blotting substrate (Bio-Rad).