TEM
An imaging-ready carbon-coated copper grid was placed on the sample and remained there for 3 min. The grid was then stained with a 2% uranyl formate aqueous solution (containing 25 mM NaOH) for 3 min and was subsequently washed two times with distilled water. TEM imaging was performed at 100 kV.
Yeast Sup35 monomer release and se mi-denaturating detergent agarose gel electrophoresis (SDD-AGE) [24]
1.2% agarose solution (40 mL) in 1 × TAE was melted in a microwave oven, and 400 µL 10% SDS was added to the gel solution. Subsequently, this mixture was carefully cast into the slab to avoid any bubbles in the gel. After the gel had set, the slab was placed in the electrophoresis chamber (Bio-Rad, Mini sub-cell) and submerged in 1 × TAE containing 0.1% SDS at 20 V for 4 h.
The release of Sup35 monomers from the 74-D694 [psi-] cells was performed using the lyticase method. Cell lysates or purified Sup35 protein (40 µL each) was immediately mixed with 6 × loading buffer (300 mM Tris pH 6.8, 60% glycerol, 0.6% bromophenol blue) and loaded into an agarose gel for SDD-AGE. Subsequently, the gels were used for western blot.