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12.シナプトゾーム単離法(清水孝雄)

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