Two-dimensional gel electrophoresis

Samples containing 100 µg protein were passively rehydrated onto 7 cm IPG strips (Bio-Rad, Hercules, CA, USA) with a pH range 4–7 for 12–16 h, followed by isoelectric focusing through the three-step protocol: the initial voltage was limited to 250 V for 10 min then increased to 3,500 V for 2,800 Vhr, then continued at 3,500 V to 3,700 Vhr. The current was limited to 50 µA per strip at 20 °C. After isoelectric focusing, the strips were first equilibrated in equilibration (EQ) buffer containing 2% 1,4-dithiothreitol (Bio-Rad, Hercules, CA, USA) for 15 min, followed by another 15 min equilibration in the EQ buffer containing 2.5% Iodoacetamide (Bio-Rad, Hercules, CA, USA) with shaking. The proteins were resolved on a 10% SDS-PAGE for the albumin-containing samples and 12% SDS-PAGE for the albumin-depleted samples. The gels were fixed in methanol/acetic acid/water solution (4:1:5) for 2 h, followed by 3 h of staining with 1X Flamingo fluorescent stain (Bio-Rad, Hercules, CA, USA).