Ipgphor 3 system

Approximately 200 μg of plasma were solubilized in the rehydration buffer containing 8 M urea, 2% CHAPS, 0.002% bromophenol blue, 2% IPG buffer pH 3–10 and 65 mM DTE. The samples were then separated by the Immobiline Drystrip on the IPGphor III System (GE Healthcare, Göteborg, Sweden) for the first dimension. The 2-DE was carried out on 10% acrylamide gels at 30 mA/gel [38 (link)]. All gels were visualized by silver staining and then scanned using an Imagescanner (GE Healthcare, Göteborg, Sweden). All experiments were repeated three times to confirm the reproducibility.

Two-dimensionl electrophoresis is a powerful tool to analyze differential protein expression levels with or without 250 μg/mL SBE treatment according to previously described procedures [50 (link)] which have been both high sensitivity and efficacy. The cell lysate (180 μg) was applied to 18-cm IPG, pH 4–7 linear strip (GE Healthcare, Uppsala, Sweden) and separated on the IPGphor III System for the first dimension. The 2-DE was carried out on 10% acrylamide gels (Bio-Rad, Hercules, CA, USA) and the gels were visualized by silver staining. Protein spots were quantified using the Prodigy SameSpots software (Nonlinear Dynamics version 1, Newcastle, UK). More than 1.5-fold alterations at 95% confidence interval (p < 0.05) were considered statistically significant. All experiments were repeated three times to confirm reproducibility.