METHODS
Differential in-gel electrophoresis (DIGE)
Different samples were labelled with Cy3 and Cy5 and crisscross experimental design was used to eleminate differences between dyes. Cultured cells were washed with DIGE wash buffer (5mM magnesium acetate, 10mM Tris, pH=8) and lysed in DIGE lysis buffer (8M urea, 4% CHAPS, 30mM Tris, pH=8.5). After centrifugation at 13,200 rpm for 20 min, supernatant was harvested and protein concentration was determined by Bradford method.
The fluorescence dye labelling reaction was carried out at a dye/protein ratio of 200pmol/50µg. After incubation on ice for 30min, the labelling reaction was stopped by scavenging non-bound dyes with 10mM lysine for 15min. Fifty micrograms whole cell extracts of each sample were labelled with fluorescent dyes (Cy3 or Cy5) and mixed with internal pool standard (labelled with Cy2).
Samples were mixed with 2× buffer (8M urea, 4% w/v CHAPS, 2% w/v DTT, 2% v/v Pharmalytes pH 3-10) and loaded on immobilized pH gradient strips (18cm, pH 3-10, nonlinear). As in traditional 2-DE, after rehydration overnight, samples were separated using the same protocols. Fluorescence images were acquired using the Typhoon variable mode imager 9400 (GE healthcare), visualized by silver staining and scanned in transmission scan mode using a calibrated scanner (GS-800, Bio-Rad) for documentation.
Gel images were analysed by DeCyder. By matching and normalising the internal standard from different DIGE gels, samples from different DIGE gels using same standard can be matched and compared, and statistical analysis can be carried on. Spots showing a statistically significant difference in intensity were excised for in-gel tryptic digestion.