Protein 2-DE Protocol for Separation

Protein 2-DE was performed as previously described [47 (link)]. A total of 1,200 μg of proteins extracted from each sample were first separated by isoelectric focusing (IEF) using gel strips with a pH gradient of 4 to 7 (Immobiline Dry Strip, pH 4–7 NL, 17 cm; BioRad, Hercules, CA, USA). The strips were rehydrated for 14 h in 320 ml of dehydration buffer and then focused at 20°C for a total of 64 kV-h with a PROTEAN IEF Cell system (Bio-Rad). After IEF, the strips were equilibrated for 20 min, first in equilibration buffer I [6 M urea, 0.375 M Tris (pH 8.8), 2% (w/v) SDS, 20% (v/v) glycerol, and 2% (w/v) DTT) and then in equilibration buffer II [6 M urea, 0.375 M Tris (pH 8.8), 2% (w/v) SDS, 20% (v/v) glycerol, and 2% (w/v) iodoacetamide]. The equilibrated strips were then placed over 12.5% (w/v) sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels for 2-DE at 25 mA for 5 h. The 2-De gels were stained with colloidal CBB.