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).