Size exclusion chromatography (SEC) of Sup35 complexes and
monomers
Freshly prepared Sup35 using an Ni2+-NTA agarose
column was dialyzed with buffer C (20 mM Tris, pH 8.0, 1.2 M NaCl) for
12 h to remove imidazole. Subsequently, the sample was injected into
a
HiLoad Superdex 200 pg 16/60 size exclusion chromatographic (SEC) column
(1 mL/min, 17-1069-01) for peak I separation (flowing phase, buffer C).
For the second SEC separation, the eluted samples were stored for 14-day
at 4oC without concentration. For SDS-PAGE and
semi-denaturating detergent agarose gel electrophoresis, the samples
were concentrated by 2500 g centrifugation with an ultrafiltration tube
(Millipore, 3000 MWCO).