CyDye labeling

The DIGE technology is an unbiased approach to identify differences in protein expression and the use of an internal standard enables identification of protein differences as small as 10% [22]. Protein extracts were labeled using the fluorescent cyanine dyes developed for 2D-DIGE technology (GE Healthcare) following manufacturer’s protocol. Protein extracts (30 μg) were labeled with 300 pmol of fluorescent dye (Cy2 or Cy3 or Cy5). Protein samples from COPD patients and healthy age-matched control were randomly labeled with Cy3 or Cy5. To exclude the effects of preferential labeling each dye was used a similar number of times in each group. An internal standard, created by pooling 15 μg of each protein sample, was labeled with Cy2. Labeling was stopped by adding lysine and equal volume of 2 × IEF buffer (8 M Urea, 2 M Thiourea, 4% CHAPS, 300 mM DTT, 1.0% IPG buffer 3-10NL, 0.004% Bromophenolblue) to each sample.