Spermatid Perinuclear Ribonucleic Acid: MATERIALS AND METHODS(2)

2 Feb

MATERIALS AND METHODS(2)Microtubule Purification from Mouse Testes

Microtubules were purified from mouse testes by a taxol-dependent method modified from Vallee. Testes were dissected from sexually mature Swiss Webster male mice, and the tunicas were removed. Approximately 400 ^g of tissue was resuspended on ice in 1 ml microtubule (MT) buffer (0.1 M Pipes, pH 6.6, 1 mM EGTA, 1 mM MgSO4) with protease inhibitors (1 ^g/ml aprotinin, 1 ^g/ml pep-statin, 3.4 ^g/ml PMSF, 10 ^g/ml soybean trypsin inhibitor, 1 ^g/ml Na-p-tosyl-L-arginine methyl ester hydrochloride, 1 ^g/ml leupeptin) and sonicated for 15-20 sec followed by incubation on ice for 15 min.

The lysate was cleared by spinning at 30 000 X g at 4°C for 15 min; this was followed by a 30-min spin at 100 000 X g at 4°C. Taxol (Sigma) was added to the supernatant at a final concentration of 20 ^m, guanosine triphosphate (GTP; Sigma) was added to a concentration of 1 mM, and the tube was placed at 37°C for 15 min. Polymerized microtubules were pelleted through a 10% sucrose cushion (prepared in MT buffer supplemented with protease inhibitors, with taxol and GTP added to 20 ^m and 1 mM concentrations, respectively) at 30 000 X g, for 15 min, at 37°C.