2D differential in gel electrophoresis (2D-DIGE): sample preparation and protein labeling

Proteins from samples were directly extracted in DIGE lysis buffer (7 M urea, 2 M thiourea, 4% CHAPS, 30 mM Tris and 1x complete protease inhibitor EDTA free, Roche) using the 2D grinding kit system (General Electric Healthcare). The solubilized proteins were separated from non-solubilized cellular components by centrifugation (20,000 g × 20 min). Salts and any interfering components were removed using the 2D Clean-up kit (GE Healthcare) and after precipitation, proteins were resolublized in DIGE label buffer (7 M urea, 2 M thiourea, 4% CHAPS, 20 mM Tris-pH 8.5). Protein concentration was determined using the Bradford Bio-Rad Protein Assay (RcDc Kit) with bovine serum albumin (BSA) as standard.

Proteins from each experimental group were randomly labeled either with Cy3 or Cy5 following to the manufacturer's instructions (GE Healthcare). Briefly, 50 μg protein of each sample was labeled with 400 pmol CyDye DIGE Fluor minimal Dye by vortexing and incubated on ice in the dark for 60 min. The labeling reaction was stopped with 1 μL of 10 mM lysine followed by incubation on ice for 10 min. An internal standard sample was prepared by pooling 25 μg of protein from each sample, and by labeling by Cy2 as described above. Differentially labeled samples (150 μg total protein) were mixed and 65 mM DTT and 1% ampholytes (pH = 3–10 NL) were added to the mixture before running the first dimension.