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.