Figure 12.
Synaptophysin biotinylated at 18°C is quantitatively extracted from paraformaldehyde-fixed PC12 cells by Triton X-100 (A) and is accessible to anti-synaptophysin after digitonin permeabilization of fixed cells (B). (A) PC12 cells were incubated without (−) or with (+) sulfo-NHS-LC–biotin for 30 min at 18°C, chased for 5 min at 18°C in the presence of glycine, and fixed (+) or not fixed (−), and Triton X-100 extracts were subjected to streptavidin–agarose adsorption. Specific immunoreactivity due to the binding of biotinylated synaptophysin to streptavidin–agarose was determined by incubating the beads without (−) or with (+) anti-synaptophysin antibody (α-Sy) followed by HRP-conjugated goat anti–mouse IgG antibody. Data are the mean of values obtained from three coverslips; bars indicate SD. (B) PC12 cells were incubated without (−) or with (+) sulfo-NHS-LC– biotin for 30 min at 18°C, chased for 5 min at 18°C in the presence of glycine, fixed, permeabilized with digitonin, incubated without (−) or with (+) anti-synaptophysin (α-Sy), and extracted with Triton X-100. The Triton extracts were subjected to streptavidin– agarose adsorption, and anti-synaptophysin bound to the beads via biotinylated synaptophysin was detected by HRP-conjugated goat anti–mouse IgG antibody. Data are the mean of values obtained from three coverslips; bars indicate SD. The lower synaptophysin immunoreactivity in B than A (compare ordinate scales) presumably reflects incomplete accessibility of the anti-synaptophysin to its epitope when added to digitonin-permeabilized fixed cells as compared with anti-synaptophysin addition after Triton X-100 extraction of synaptophysin and its adsorption to streptavidin–agarose beads.