Trans blot turbo rapid western blotting transfer system

For further analysis of lipid raft purity, we performed immunoblotting using different antibodies directed against lipid raft and non-raft proteins. First, equal amount of protein extracts were electrophoresed in SDS-PAGE gels, and transferred to polyvinyl (PVDF) membranes using the Trans-Blot Turbo rapid western blotting transfer system (Bio-Rad, Madrid, Spain). The membranes were incubated overnight at 4°C with the different primary antibodies used in this study: Mouse monoclonal anti-NeuN neuronal biomarker antibody diluted 1: 2,000; mouse monoclonal anti-PrP antibody diluted 1: 500; mouse monoclonal anti-GAPDH antibody diluted 1: 5,000; and rabbit polyclonal anti-flotillin 1 antibody diluted 1: 1,000). All antibodies were diluted in BLOTTO (5% non-fat dried milk in TBS). Following antibody incubation, membranes were washed three times for 5 min in TBS with 0.1% Tween-20. Reaction with peroxidase-conjugated anti-mouse or anti-rabbit secondary antibody was performed diluting the antibodies 1: 5,000 in BLOTTO for 1 h at room temperature. Specific bands were developed with Clarity^TM Western ECL Substrate and processed using Chemie-Doc MP Imaging System (Bio-Rad). The optical density was analyzed using Image Lab software.

Proteins resolved by SDS-PAGE were transferred to polyvinyl (PVDF) membranes using the Trans-Blot Turbo rapid western blotting transfer system (Bio-Rad, Madrid, Spain). PVDF membranes were blocked with 5% BLOTTO (non-fat dried milk in TBS plus 0.1 Tween-20). Then, membranes were incubated overnight at 4°C with the different primary antibodies (mouse monoclonal anti-aggregated α-synuclein (5G4 clone), anti-α-synuclein (4D6 clone), anti-PrPc, anti-Aβ peptide, and anti-APP antibodies; the rabbit polyclonal anti-pSer129 α-syn and anti-Flotillin-1 antibodies; and the rabbit monoclonal (anti-α-synuclein [EPR20535] antibody). All antibodies were diluted 1:1,000 in BLOTTO, except antibodies purchased from Santa Cruz biotech that were diluted 1:200 according to manufacturer’s indications. Membranes were washed three times for 5 min in TBS with 0.1% Tween-20. Proteins were detected using the corresponding peroxidase-conjugated anti-mouse or anti-rabbit secondary antibody diluted 1:5,000 for 1h at room temperature. Specific bands were developed with Clarity^TM Western ECL Substrate and processed using Chemie-Doc MP Imaging System (Bio-Rad). The optical density was analyzed using Image Lab software.