2.6. D-DIGE

Protein samples were loaded onto 24-cm Immobiline DryStrips, with a 3–10 nonlinear gradient pH range (GE Healthcare, Uppsala, Sweden), and rehydrated for 12 h. In the first dimension of electrophoresis, the proteins were separated according to their isoelectric point using an Ettan IPGphor apparatus (GE Healthcare, Uppsala, Sweden) at 20 °C with current limited to 50 µA per strip and the following voltage program: 500 V over 2 h, a linear gradient to 1000 V over 1 h and a linear gradient to 10,000 V over 3 h, then at 10,000 V constant for 4 h. Subsequently, the strips were equilibrated in SDS equilibration buffer (6 M urea, 75 mM Tris-HCl, pH 8.8, 29.3% glycerol, 2% sodium dodecyl sulfate, 0.002% bromophenol blue) containing 65 mM DDT for 15 min, and then in SDS equilibration buffer containing 135 mM iodoacetamide for 15 min. The equilibrated strips were then transferred to precast DIGE gels Ettan DALT Gel 12.5 (25.5 × 19.6 cm, 1 mm thickness, GE Healthcare, Uppsala, Sweden) and sealed with 0.5% agarose. A second dimension of electrophoresis was then performed at 1.5 W/gel in an Ettan Dalt-Six apparatus (GE Healthcare, Uppsala, Sweden) for 16 h. The gels were scanned using a Typhoon 9500 FLA scanner (GE Healthcare, Uppsala, Sweden) and the obtained gel images were subjected to a statistical analysis.