FIGURE 2.
Tris/Tricine SDS-PAGE and Western blot. Lyophilized α-synuclein (α-syn) was dissolved to a final concentration of 100 μm in Buffer A filtered through a 0.2-μm filter. For probing with syn-10H scFv, the sample was incubated at 37 °C for 7 days without shaking. An aliquot (20 μl) of incubated solution was run on 10% polyacrylamide Tris-Tricine SDS-PAGE followed by silver staining (A). The sample was then transferred to nitrocellulose membrane (B1) and probed with 0.3 mg/ml scFv (syn-10H) antibody. An aliquot (50 μm) of monomeric α-synuclein (time 0) was transferred to nitrocellulose membrane and probed with 0.3 mg/ml syn-10H and D10 scFv (C). Bound antibodies were detected after a 24-h incubation at 4 °C, using a 1:500 dilution of 9E10 antibody, followed by a 1-h incubation with 1:1000 dilution of anti-mouse HRP followed by staining with 3,3′-diaminobenzidine tetrachloride (Sigma). Western blot analysis of oligomeric α-synuclein probed with 0.3 mg/ml D5 scFv was reproduced from Emadi et al. (32) (B2). MW, molecular weight.