Spermatid Perinuclear Ribonucleic Acid: MATERIALS AND METHODS(3)

3 Feb

The microtubule pellet was rinsed in MT buffer and resuspended in 125 MT buffer supplemented with taxol (20 ^m final concentration), GTP (1 mM final concentration), and protease inhibitors (as above). The tube was placed at 37°C for 15 min, and microtubules were pelleted as described above. The pellet was resuspended in MT buffer and subjected to another round of purification (as above). The third microtubule pellet was resuspended in MT buffer containing protease inhibitors. Aliquots of the starting extract, the supernatants, and the microtubule pellets were analyzed by SDS-PAGE followed by Coomassie staining or Western analysis. ventolin inhaler

B inding of His-SPNR to Mouse Testis and Bovine Bra in M icrotubules

The entire protein coding region of the Spnr was cloned in-frame with the N-terminal His-tag present in the pET15b vector (Novagen, Madison, WI). A 200-ml culture of pLysS Escherichia coli (Novagen) transformed with the His-SPNR vector was grown at 37°C to an OD600 of 0.5. Cells were induced with 1 mM isopropylthiogalactoside for 1 h and harvested by low-speed centrifugation. The bacterial pellet was resuspended in 2 ml MT buffer (0.1 M Pipes, pH 6.6, 1 mM EGTA, 1 mM MgSO4) supplemented with protease inhibitors as described above. The extract was sonicated on ice and spun at 4°C for 30 min at 100 000 Xg. The supernatant was recovered and used for all subsequent binding experiments.