)Supernatant was
transferred to a 13.2 mL thinwall ultraclear centrifuge tube (Beckman-
Coulter 344059) and layered with a gradient of 0.27 M sucrose,
0.135 M sucrose and Top solution buffer (25 mM Tris–HCl; 1 mM
EDTA; 1 mM EGTA; protease inhibitor cocktail)