Fig. 1.
The effect of time of pre-electrophoretic heating at 50 °C on non-reducing SDS-PAGE analyses of h2E2 anti-cocaine mAb fragments. Panel A (top) – results using F(ab’)2, Panel B (bottom, left) – results using Fab, Panel B (bottom, right) – results using Fc. A 1.5 mm thick, 7% acrylamide gel was loaded with 2 μg (10 μL) of antibody fragment per well, electrophoresed until the tracking dye reached the bottom of the gel, and stained with Coomassie blue. The entire gels are shown. Pre-electrophoretic incubation in SDS-PAGE non-reducing sample buffer was carried out for the indicated times at 50 °C. The samples in the lanes labeled “boil” were boiled for 3 min prior to electrophoresis. In Panel A, discrete bands/folded states noted for the F(ab’)2 fragment are labeled “A”, “B”, and “C”. See Fig. 4A for the hypothesized identification of these 3 bands/differentially folded states.