In vitro Assay of Clostridial Neurotoxins

The procedure for the preparation of synaptosomes and small synaptic vesicles (SSV) from rat brain cortex follows established methods with minor modifications (Schiavo and Montecucco, 1995). 

Reactions are stopped by addition of 2% in SDS, mercaptoethanol, 5mM EDTA, 5% glycerol and boiling. 

Proteins are analyzed in SDS-PAGE and stained with Coomas-sie Blue or, after transfer onto nitrocellulose membrane, by Western Blotting with antibodies against VAMP/synaptobrevin (for TeTx, BoNT/B, D, F and G) or SNAP-25 (for BoNT/A, C and E ) or syntaxin (for BoNT/C) (see Table 2). 

Quantitative analysis of the proteolytic cleavage is performed by densitometric analysis. 

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