Rapid isolation and cryo‐EM characterization of synaptic vesicles from mammalian brain

Synaptic vesicles (SVs) store and release neurotransmitters at chemical synapses. Precise regulation of SV trafficking, exocytosis and endocytosis is crucial for neural transmission. Biochemical characterization of SVs, which is essential for research into neurotransmitter uptake and release, requires effective in vitro isolation methods. Here, we describe an improved and simple purification protocol for isolating SVs from mouse brain within 6 h, achieving a yield of approximately 0.4 mg of SVs per single brain. The use of track‐etch membrane filtration and iodixanol cushion ensured the uniform morphology of SVs and low contaminants in the sample. Cryo‐electron microscopy was used to show that the in vitro isolated SVs retained intact membrane‐associated proteins, and observation of SVs in hippocampal neurons using cryo‐electron tomography confirmed the abundance of protein coating. Thus, our protocol allows effective isolation of SVs from small volumes of mammalian brain tissue, and the properties of the isolated SVs are close to those in vivo, making them suitable for biochemical analysis.