Anti mouse αsyn monoclonal antibody

Brain tissues (1:10, w/v) were homogenized in lysis buffer (10 mM Tris-Cl, 1 mM EDTA, 0.5 mM EGTA, 140 mM NaCl, pH 8.0) containing Protease Inhibition Cocktail III. Soluble and insoluble αSyn proteins were subsequently lysed with two different detergents. The soluble part was first extracted with 1% Triton X-100 in the lysis buffer. The insoluble part in the remaining pellet was further extracted with lysis buffer containing 1% Triton X-100 /2% SDS. Protein concentration was determined by BCA Protein Assay kit (Thermo Fisher Scientific). Pre-casted 4–12% gradient gel (Invitrogen) was used for protein separation. The proteins on the gels were transferred onto 0.45μm PVDF membranes. The membrane was blocked in 5% milk in PBS for 20 min at room temperature. Anti-mouse αSyn monoclonal antibody (1:500 in 3% milk/PBS, Novus) was incubated overnight at 4°C followed by goat-anti-mouse IgG-HRP (1:1500 in 1% milk/PBS, Cell signaling) incubation for 1 hr at room temperature. An ECL detection system (SuperSignal™ West Dura, Thermo Fisher Scientific) was applied for signal detection. Anti β-actin monoclonal antibody (1:5000, Sigma) was used as a loading control.

The following were from commercial sources: Anti-β-actin monoclonal antibody (Sigma, St. Louis, MO). SuperSignal™ West Dura (ThermoFisher Scientific, Rockford, IL). Anti-mouse αSyn monoclonal antibody (Novus Biologicals, Littleton, CO). Anti-mouse αSyn monoclonal antibody (Abcam, Cambridge, MA). Goat anti-mouse Alex 595 (Invitrogen, Grand Island, NY). Goat-anti-mouse IgG-HRP (Cell signaling, Danvers, MA). Tyramide Signal Amplification (TSA) kit (Perkin Elmer, Waltham, MA). C16-, C18- and C24:1-GCs and C8-GC (Avanti Polar Lipids, Inc., Alabaster, AL).