原法 J. Cell Biol. 83, 308-318, 1979
1. All procedures should be carried out at below 4 degree C unless stated otherwise.
2. Preparation of P2 fraction.
Rat brain 20 g/20 rats (cerebral cortex only)
Wash 3 times with solution A (0.32 M sucrose/3 mM HEPES, pH 7.3).
Homogenize 12 up-down strokes with a Teflon-glass homogenizer at 800-900 rpm in 4 volumes of Solution A.
Dilute to 10% (w/v) with Solution A.
Centrifuge at 1,475 x g, for 10 min.
Supernatant
Centrifuge at 17,300 x g for 10 min.
Pellet (P2 fraction)
Resuspend in Solution A. (amount appropriate for adjusting tube volume)
Centrifuge at 17,300 x g for 10 min.
Pellet (Washed P2 fraction)
Suspend in Solution A (eq. 2 ml/g cortex)
3. Sucrose density gradien
Hitachi 28SA rotor 6 tubes
4. Synaptic membrane
Crude synaptosome is recovered by aspiration (25 ml) from
Dilute with 4 volumes of Solution A (ca. 100 ml)
Centrifuge at 37,800 x g for 20 min.
Pellet (Synaptosome)
Lyse by suspending 10 ml of 6 mM Tris-HCl, pH 8.1/g cortex using a Potter homogenizer (osmotic shock)
Stir on ice for 45 min.
Centrifuge at 32,800 x g for 20 min.
Pellet (synaptic membrane)
5. Synaptic vesicle
Sup
Centrifuge at 78,000 x g for 120 min
Pellet (Crude synaptic vesicle pellet)
Suspend in Solution A (5 ml)
Sucrose density gradient
Hitachi RPS 40Ti
Dilute with 4 volumes of Solution A
Centrifuge at 78,000 x g for 60 min
Pellet (Synaptic vesicle pellet)
Modifications:
Hunter et al. J. Cell Biol. 96, 1374-1388, 1983
Scaife and Margolis J. Cell Biol. 111, 3023-3033, 1990
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