Alpha-synuclein deficient knockout mice were generated.
Mouse brains were analyzed by Western Blot using polyclonal anti-synuclein which recognizes both alpha and beta. Total mouse protein was run on two dimentional electrophoresis using pH 4-8 ampholines and 10% PAGE gel and then Western blotted using anti-alpha-synuclein (Transduction Labs.) with detection by HPR and chemiluminescence (Amersham Biosciences).
Synaptosomal protein or post nuclear supernantant from cultured hippocampal neurons were analyzed by Western blot using the following antibodies: anti-synapsin I, anti-synaptophysin, anti-synaptotagmin, anti-rab3a, and anti-amphiphysin (Stressgen); anti-VAMP-2 (vesicle-associated membrane protein-2)and anti-SNAP-25 (synaptosomal-associated protein) from Santa Cruz; Anti-flotillin from Transduction Labs and anti-SV2 (ATCC). Quantitating Western blot was done by making a standard curve with recombinant alpha-synuclein, electrophoresing it on SDS-PAGE and immunoblotting with anti-synuclein 202 (Zymed). The bands were scanned by densitometry and quantitated using ImageQuant software (Molecular Dynamics).