Analyzing Yeast Protein Aggregation

Yeast samples were collected and cell density was determined at A₆₀₀. Identical number of cells were collected by centrifugation (18,000 x g, 1 minute, 4°C), washed in 10 mM NaN₃ in PBS, and frozen (-20°C), to allow simultaneous processing. Cells were lysed in 0.4 ml /2 A₆₀₀ of lysis buffer (0.2 M NaOH / 71 mM β-mercaptoethanol) followed by incubation on ice for 30 minutes, adjustment to pH 7 with HCl, and boiling, as described [20 (link)]. Boiled lysates were adsorbed to, or filtered through 0.2 μm nitrocellulose membranes, in the absence or presence of 2% SDS, respectively. PolyQ proteins and actin were detected by immunoblotting with rabbit anti-GFP antibody (ab290, Abcam) and mouse anti-actin antibody (ab3280, Abcam), respectively, followed by DyLight 680-labled goat anti-rabbit IgG (072-06-15-06, KPL) and IRDye 800CW-conjugated goat anti-mouse antibody IgG (LI-COR Biosciences), respectively. Secondary antibodies were visualized by the Odyssey Infrared Imaging System (LI-COR Biosciences) and quantified by the Odyssey software (v 3.0). Aggregation Indexes were calculated, as described [20 (link)].