Pdquest advanced software v 8

The 2-DE images from gels stained with Pro-Q Diamond and SYPRO Ruby fluorescent dyes were captured with the Gel Doc XR+ Imaging System (Bio-Rad Laboratories). Analysis of digitalized gel images was performed with PDQuest Advanced software v. 8.0.1 (Bio-Rad Laboratories). Protein volumes of detected and matched spots over biological replicates were measured following subtraction of background noise and total valid spot normalization. Automatic spot analysis by PDQuest software was manually validated. Only protein spots reproducibly detected in at the least two of four biological replicates were selected for image analyses. The observed isoelectric point (pI) value of protein spots was determined from their gel position relative to focused strips of linear pH gradient, whereas molecular mass markers ranging from 15 to 200 kDa (Fermentas, Ontario) were used to assess the observed molecular mass (M_r). Protein fragments were identified by comparing the M_r observed on 2-DE gels with the theoretical M_r of the full-length sequence and they were excluded from further analysis.