Differential centrifugation

The homogenate was distributed in 1.5 ml tubes at 1 ml per tube, and pellets collected at 3,000 x g for 10 min, 9,000 x g for 15 min, and 100,000 x g for one hour, were named nuclear, mitochondrial and microsomal, respectively. The supernatant from the last centrifugation was named the cytosol [35,36]. Open-top thin-wall polypropylene tubes, 14 x 89 mm, were used for all ultracentrifugation steps at velocities ≥ 100,000 x g. Pellets were solubilized in isoelectric focusing buffer (IEF, 7 M urea, 2 M thiourea, 2% [w/v] CHAPS, 40 mM dithiothreitol) for characterization of differential centrifugation. Otherwise, pellets were stored on ice for their further separation in sucrose gradients.