Bio gel p 30 gel

Conjugations of primary antibodies and NeutrAvidin to AlexaFluor647 was carried in 100–120 μl volume containing 100 mM sodium bicarbonate with 1mg/ml final concentration of antibody and a ~10× molar excess of reactive AF647 to achieve a final ratio of 2–5 dye molecules per antibody. The reaction was carried out for 1 hour at room temperature with constant mixing, protected from light. Conjugations of primary antibodies to PC-biotin were carried in 100–120μl volume with 1 mg/ml final concentration of antibody and a 25 × molar excess of PC-biotin-NHS, and 100mM sodium bicarbonate. The reaction was carried out for approximately one hour at room temperature with constant mixing and protected from light.
Labeled antibodies and NeutrAvidin were purified using gravity columns with Bio-Gel P-30 gel (BioRad #150–4154) to separate antibody from free dye/label. For PC-biotin-conjugated antibody, which is not visible within the column, multiple fractions were collected, and protein concentration was measured using 280 nm-absorbance to determine the fractions that contain antibody. Protein concentration was confirmed by BCA assay (Pierce). Conjugation of AF647 to antibodies containing BSA and/or other carrier proteins was done using the APEX AlexaFluor-647 antibody labeling kit according to the manufacturer instructions.

For microscopic detection of aggregates, we used fluorescent antibodies. The mouse anti-aSyn monoclonal antibody 211 (Santa Cruz Biotechnology, Inc., Dallas, USA) was labeled with CF633 (Biotium, Freemont, USA), whereas the anti-tau Tau5 antibody (Biolegend, San Diego, USA) was labeled with CF488A (Biotium, Freemont, USA). The labeling process was performed as described in the manufacturer’s protocol. The dyes were activated as succinimidyl esters to react covalently with the amines of the antibody in carbonate buffer. For purification of each labeled antibody, a polyacrylamide bead suspension (Bio-Gel P-30 Gel, Bio-Rad Laboratories, Inc., Hercules, USA) was used. The concentration and the degree of labeling was determined according to the manufacturer’s protocol.