Subfractionation of Synaptosomes

Synaptosomes can be subfractionated into a heavy membrane fraction that contains plasma membranes, synaptic vesicle clusters and most of the contaminating membranes, a light membrane fraction that is enriched in synaptic vesicles and devoid of measurable contamination by mitochondria or neuronal plasma membranes, and a 

To osmotically lyse the synaptosomes, 2.7 ml of ice-cold distilled water are added, followed by rapid homogenization in a glass teflon homogenizer (6 strokes at 2000 r.p.m). 

The remaining supernatant is centrifugued at 100 000 g for 1 h (shorter times may be used if small rotors, e.g., Beckman TIa 100.3, are used) yielding a pellet enriched in synaptic vesicles, and a cytosol fraction in the supernatant. 

Breakdown of the substrates can be checked by SDS-PAGE and immunoblotting. Monoclonal antibodies for syntaxin 1 a and 1 b, as well as for SNAP-25 are available from commercial sources (Sigma, Stern-berger Monoclonals). Antibodies for synaptobrevin have been raised by many laboratories and can usually be obtained from one of them. 

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