Protein extraction and sample preparation for two-dimensional gel electrophoresis (2D-E)

Protein extraction and sample preparation for 2D-E were performed as reported in Silvestri et al. (2006). Liver tissue was suspended in sample buffer [20 mM Tris, 7 M urea, 2 M thiourea, 4% CHAPS, 10 mM 1,4-dithioerythritol (DTE), 1 mM EDTA, and a mixture of protease and phosphatase inhibitors]. The suspensions were homogenized using a Polytron homogenizer, sonicated for 30 s, and centrifuged at 150,000 g for 45 min. The obtained supernatants contained the total liver proteins solubilized in the isoelectrofocusing (IEF)-compatible agents. The protein content of each sample was determined by Bio-Rad's DC method (Bio-Rad Laboratories, Hercules, CA). Total protein extracts were prepared for each animal, and each individual was assessed separately.