| Note |
The tissue was dissected into small pieces and homogenized with 10 up-and-down, unrotated strokes in forty volumes of cold hypotonic buffer (1.5 mM MgCl2, 10 mM NaCl, 10 mM Tris-HCl (pH 7.4, 1/100 volume protease inhibitor cocktail, 1/100 volume PMSF) per brain weight. After 30 min on ice, samples were homogenized with 20 up-and-down strokes and disrupted by cavitation at 700 psi for 15 min at 4 ºC in a pressure vessel. The homogenates were centrifuged at 10,000 × g for 10 min at 4 ºC, and the supernatants were centrifuged again at 10,000 × g for 10 min at 4ºC. The resulting supernatants were collected as cytosol fraction. Each pellet was suspended in suspension buffer (10 mM Tris-HCl (pH 7.4), 50 mM sucrose), layered on top of a 38% (w/v) sucrose solution, and centrifuged at 100,000g for 40 min at 4 ºC. The turbid layer at the interface was recovered and centrifuged at 100,000g for 40 min at 4 ºC. The plasma membrane fraction was obtained from the resulting pellet, and resuspended in suspension buffer. |