Biochemical Characterization of Brain Fractions

For biochemical characterization of fractionated human brain tissue, 5μg of lysate from either the HS soluble or Triton X-100 insoluble fractions were loaded onto 15% polyacrylamide gels and resolved by SDS-PAGE, followed by electrophoretic transfer onto 0.2μm pore size nitrocellulose membranes (Bio-Rad, Hercules, CA) in carbonate transfer buffer (10 mM NaHCO₃, 3 mM Na₂CO₃, pH 9.9, 20% methanol) [17 (link)]. Membranes were blocked in 5% dry milk/Tris buffered saline (TBS) and incubated overnight at 4°C with anti-αSyn antibody 3H11 [14 (link)] diluted in block solution. After washing in TBS, membranes were incubated with goat anti-mouse secondary antibody conjugated to horseradish peroxidase (Jackson Immuno Research Labs, Westgrove, PA) diluted in 5% dry milk/TBS for 1 hour at room temperature; immunocomplexes were detected using Western Lightning-Plus ECL reagents (PerkinElmer, Waltham, MA) followed by chemiluminescence imaging (PXi, Syngene, Frederick, MD).