2-DE Gels of Pecten maximus gills at three different temperatures and two O2 levels (normoxia or hypoxia)

IPG strips (pH 4–7, 13 cm; GE Healthcare) were passively rehydrated with 250 µl of protein solution in wells for 14 h. Isoelectric focusing was conducted using the following protocol: 250 V for 15 min, 500 V for 2 h, gradient voltage increased to 1 000 V for 1 h, gradient voltage increased to 8 000 V for 2,5 h, 8 000 V for 3 h, and finally reduced to 500 V (Ettan IPGphor3, GE Healthcare). To prepare for the second-dimension SDS-PAGE, strips were incubated in equilibration buffer (50 mM Tris-HCl pH 8.8, 6 M urea, 30% glycerol, 2% SDS and 0.002% Bromophenol Blue) for two 15 min periods, first with 1 g.l-1 dithiothreitol and then with 48 g.l-1 iodoacetamide. IPG strips were placed on top of 12% polyacrylamide gels, which were run in thermo-regulated electrophorese unit at 10°C (SE 600 Ruby, Amersham Biosciences) at 10 mA per gel for 1 h and then 30 mA per gel until complete migration. Gels were subsequently stained with “Blue Silver” (Candiano et al., 2004) and destained with Milli-Q water for 48 h. The resulting gels were scanned with a transparency scanner (Epson Perfection V700) in gray scale with 16-bit depth and a resolution of 400 dpi.