Purification of 26S Proteasome Complex

ATP and glycerol were added to buffers all along the procedure to preserve the interactions between the 20S core and 19S regulatory particles and therefore to maintain 26S proteasome stability [22 (link)]. The 26S proteasome purification was performed as previously reported [18 (link)] with slightly modifications. The active fractions recovered after the chromatography on Phenyl Sepharose column were dialyzed against 25 mM Tris-HCl, pH 7.5 and loaded onto a Superose 6 PC 3.2/30 column (Pharmacia Biotech, Pittsburgh, PA, USA) connected to a SMART System (Pharmacia Biotech). The elution buffer was 25 mM Tris-HCl, pH 7.5 added with 50 mM NaCl and the flow rate was 0.1 mL·min⁻¹. Active fractions were pooled and the purified proteasome was stored in 25 mM Tris-HCl pH 7.5, 1 mM ATP and 10% glycerol.