For the first dimension, electrophoresis was applied onto IPG strips of 18 cm, pH=4–7 (Bio-Rad, Hercules, CA) rehydrated by loading the samples diluted with rehydration buffer (8M urea, 4% CHAPS, 2% ampholyte, 50 mM DTT, and traces of bromophenol blue) overnight. After the gel rehydration, IEF was performed at 300V/ 1h, 500V/ 1h, 1000V/ 2h, and 3500V/ 12h using a Multiphor II electrophoresis unit and EPS 3500 XL power supply (Amersham, Piscataway, NJ) (26 (link)).
For the protein to be transferred from the first to the second dimension, IPG strips were incubated in an equilibration solution (50 mM Tris- HCl, pH= 8.8, 6M urea, 20% glycerol) (Merck, Germany), 2% SDS (Sigma), and 0.01% bromophenol blue (Merck, Germany) containing 2% DTT for 15 minutes and then re- incubated in the equilibration solution containing 2.5% iodoacetamide (Merck, Germany) for 15 minutes. Strips were placed on top of 10–15% gradient SDS-PAGE and sealed with agarose solution (Bio- Rad) (0.5% agarose plus a few grains of bromophenol blue).
The 2DE was carried out at 16 mA/gel and 24 mA/gel at 20 °C for 30 minutes until the dye front reached the bottom of the gel (24 , 26 (link), 27 (link)). Gels were silver- stained under the same conditions by freshly prepared silver reagents.
For the protein to be transferred from the first to the second dimension, IPG strips were incubated in an equilibration solution (50 mM Tris- HCl, pH= 8.8, 6M urea, 20% glycerol) (Merck, Germany), 2% SDS (Sigma), and 0.01% bromophenol blue (Merck, Germany) containing 2% DTT for 15 minutes and then re- incubated in the equilibration solution containing 2.5% iodoacetamide (Merck, Germany) for 15 minutes. Strips were placed on top of 10–15% gradient SDS-PAGE and sealed with agarose solution (Bio- Rad) (0.5% agarose plus a few grains of bromophenol blue).
The 2DE was carried out at 16 mA/gel and 24 mA/gel at 20 °C for 30 minutes until the dye front reached the bottom of the gel (24 , 26 (link), 27 (link)). Gels were silver- stained under the same conditions by freshly prepared silver reagents.