Brain Homogenate Fractionation and Preservation

This method has been previously described (Theunis et al., 2013 (link)). Briefly, total brain homogenate was centrifuged for 30 min at 150,000 g at 4°C in 2 mL Beckman tubes. The supernatant (S1) was separated from the pellet and stored at −80°C. The pellet was further re-suspended in 500 μL of sucrose buffer (10 mM Tris–HCl (pH 7.4), 0.8 M NaCl, 10% sucrose, 1 mM EGTA, and 1 mM PMSF) and centrifuged for 10 min at 14,000 g at 4°C. The soluble fraction was then incubated for 1 h at room temperature in 1% sarkosyl and then centrifuged for 30 min at 150,000 g at 4°C. The supernatant (sarkosyl soluble fraction) was collected; the pellet (sarkosyl insoluble fraction) was then re-suspended in 50 μL of TBS buffer (10 mM Tris HCl (pH 7.4), 0.8 M NaCl) and both fractions were stored at −80°C until analysis.