Antibody Selectivity for α-Syn Aggregate versus Monomer

In DDI water, α-syn monomer (1 mg/mL; Abcam, ab51189) and α-syn aggregate (2 mg/mL; Abcam, ab218817) were each used to prepare separate dilution series containing the following amounts of protein: 350, 289, 144.5, 72.3, 36.1, 18.1, and 9.03 ng/μL. Of each sample, 2 μL was spotted in duplicate onto two different methanol-activated PVDF membranes, affording the following amounts of α-syn monomer or α-syn aggregate on the membranes in each dot: 700, 578, 289, 144, 72.3, 36.1, and 18.1 ng. Using the procedures described above, one membrane was immunostained using MJFR-14 anti-aggregate α-syn primary antibody (Abcam, ab209538; 1:10,000 dilution) followed by IRDye 800CW donkey anti-rabbit IgG secondary antibody (LI-COR, 926-32213; 1:20,000 dilution). The other membrane was immunostained using 5G4 anti-aggregate primary antibody (Millipore Sigma, MABN389; 1:4000 dilution) and IRDye 800CW donkey anti-mouse IgG secondary antibody (LI-COR, 926-32212; 1:20,000 dilution). The membranes were visualized using an Odyssey CLx imager (LI-COR) on the 800 nm channel. Blots were quantified using Image Studio data analysis software (LI-COR) to analyze relative aggregation of α-syn. Prism was used for further data processing.