Characterization of Yeast Protein Aggregates

Protein extracts were electrophoresed under reducing conditions on SDS-PAGE minigels and electroblotted onto a PVDF membrane (Amersham Pharmacia Biotech). Protein concentrations were measured using a NanoDrop ND-8000 spectrophotometer. Primary antibodies used were Sup35 [35 (link)], GFP (Invitrogen, UK), Pgk1 (ThermoFisher Scientific, UK), and Hsp104 (Abcam, UK). Insoluble protein aggregates were isolated as previously described [36 (link)]. Aggregated proteins were visualized by silver staining with the Bio-Rad silver stain plus kit. The analysis of Sup35 amyloid polymers by semi-denaturing detergent agarose gel electrophoresis (SDD-AGE) was performed as described previously [37 (link)].