Comparative Proteomic Analysis: 2D-DIGE

One-dimensional analytical gel electrophoresis was performed using Immobiline Dry Strips (24 cm, pH 3-11; GE Healthcare, Sweden) and then the IPG strips were equilibrated, first with dithiothreitol (DTT) (15 min, RT, gentle stirring, 5 mM Tris–HCl, pH 8.8, 6M urea, 30% glycerol, 2% SDS 65 mM). Strips were then equilibrated for 15 min in the same solution containing 250 mM iodoacetamide (IAA). followed by second-dimension sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) performed on 12.5% fixed gels using an Ettan Dalt Six device (GE Healthcare, Sweden) were carried out as previously described by Alfadda et al. [77 (link)]. Further, the 2D-DIGE gels were scanned on the Typhoon 9410 scanner using excitation/emission wavelengths specific for Cy2 (488/520 nm), Cy3 (532/580 nm), and Cy5 (633/670 nm).