Pdquest system

The scanned images of the silver-stained 2D gels and of the visualized Western blot membranes were input to a PDQuest system (BioRad, version 7.1, Hercules, CA) to generate the synthetic image that contained the Gaussian spots (Gaussian image) with a defined volume (volume = optical density (OD) × width (mm) × length (mm)) and quality [52 (link)]. All subsequent spot-matching and analysis steps were performed on the Gaussian spots. In order to minimize the effect of any experimental factor on a spot volume, each spot volume was normalized to the total optical density in each gel image [52 (link)].

First-dimension, IEF, was carried out on a Mulitphor II instrument (GE Health) with 70 μg protein sample and precast IPG strips (pH 3–10 NL; 180 × 3 × 0.5 mm). After equilibration of IEF-separated proteins, second-dimension, SDS-PAGE, was carried out with a 12% PAGE resolving gel (190 × 205 × 1.0 mm) in a vertical PROTEAN plus Dodeca™ Cell (Bio-Rad) which can analyze up to 12 gels at a time. 2DGE-separated proteins were visualized with a modified silver-staining method. Silver-stained 2DGE gels were digitized and analyzed with a PDQuest system (Version 7.1.0; Bio-Rad). A matched analysis set that contained 30 gel images from 8 control pituitary samples, 9 gel images from 3 NF-NFPA samples, 9 gel images from 3 LH-NFPA samples, 9 gel images from 3 FSH-NFPA samples, and 9 gel images from 3 LH/FSH-NFPA samples (a control pituitary as master gel) used to compare each DEP of NF-, LH-, FSH-, and LH/FSH-NFPAs relative to controls, respectively. Comparative analyses were carried out with the mean normalized volume between each NFPA subtype and controls. The “cutoff point” value for a significant difference of a differential spot was a three-fold difference. The detailed 2DGE method and 2D gel image analysis were described [11 (link)].