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Before running one-dimensional gel, protein aliquots of 20 µg each were mixed with loading buffer (60 mM Tris-HCl pH 6.8, 2% SDS, 10% glycerol, 5% β-mercaptoethanol, 0.01% bromophenol blue), and denatured at 100°C, 5 min. For protein separation, 18 cm denaturing polyacrylamide gel was prepared with 4% stacking and 13% running parts. Protein fractionation was performed in Protean II xi Cell (Bio-Rad) with running buffer (25 mM Tris, 192 mM glycine and 0.1% SDS) at 10 mA per gel for 1 h and 30 mA per gel until tracking dye disappeared, approximately 5 h. Gels were stained by sensitive colloidal Coomassie G-250. Images were digitalised with a resolution of 300 dpi and 16-bit grayscale pixel depth on Umax ImageScanner (GE Healthcare).

The protein samples were separated by 1 dimensional- and 2 dimensional- polyacrylamide gel electrophoresis (1D- and 2D-PAGE). For 1D-PAGE, the protein samples (10 μg) were separated by 12.5% polyacrylamide gels as described by Laemmli [44 (link)]. For 2D-PAGE, equal amounts of protein (60 μg) from each sample were separated as follows. In the first dimension, IPG strips (GE Healthcare, USA) that were 7 cm in length and pH 3–10 were used. Electrophoresis was performed following the specific conditions described by the manufacturer. After isoelectric focusing (IEF), the proteins were separated by SDS-PAGE in the second dimension using 12.5% polyacrylamide gels. The gels were stained using the Coomassie Blue-G staining method. For each biological replicate, one set of high-resolution gels, run at different times, was selected for further analysis. The relative abundance of each protein spot was quantified. The gel images were scanned by a UMAX image scanner coupled with Labscan software (GE Healthcare, USA). For the 2D-PAGE profile, protein spots were detected, matched, and volume-quantified using ImageMaster 2D platinum v.6.0 software (GE Healthcare, USA).