Protocol for single-molecule analysis of synaptic protein complex-mediated vesicle recruitment

Single-molecule pull-down (SIM-Pull) combined with total internal reflection fluorescence (TIRF) microscopy enables direct visualization of proteins and multi-protein complexes. Here, we present an extended SIM-Pull protocol for analyzing protein interactions at the active zone and their ability to recruit isolated synaptic vesicles (SVs). We describe steps for visualizing and quantifying SV recruitment mediated by STX1A-SNARE and RIM1-Rab3a interactions. This technique allows the examination of subcellular vesicle-associated protein-protein interactions at a molecular level in a near-native cellular context.