NROC69H3 Chapter Notes -Epitope, Sted Microscopy, Stimulated Emission
Document Summary
Neurochemical transmission: nt released by exocitosis, vesicle membrane retrieved by endocytosis -> vesicle regenerate and refill. Synaptotagmin i - protein resident in the vesicle membrane. Full fusion: synaptic vesicle membrane undergoes exocytosis by collapsing into the plasma membrane, involves mixing and lateral diffusion of vesicle proteins across plasma memb before they are re-internalized by endocytosis. Proteins need to be sorted at plasma memb and or in an endosomal intermediate to regenerate vesicle. If vesicles constituents stay patched together after exocytosis, the recycling machinery would just have to internalize the fused vesicular patch without sorting. Between synaptotagmin pool remaining on surface of the plasma membrane & that internalized by endocytosis -> 2 protocols. Labelling performed on ice, in absence of ca2+ Labelling performed at 37 degrees c allow for uptake. Antibody binding to both surface-exposed and internalized synaptotagmin pools: to selectively label internalized pool -> blocked surface-bound antibodies with unlabelled secondary antibodies before adding dye-labelled secondary antibodies.
