Two-Dimensional Electrophoresis (2-DE) Workflow

Two-dimensional electrophoresis (2-DE) procedure was carried out as previously described^{40 (link)}. Samples were solubilized in rehydration solution (8 M urea, 2 M thiourea, 4% (w/v) CHAPS, 1% (v/v) Triton X-100, 10 mM Tris, 65 mM DTT, 0.8% (v/v) IPG buffer (pH 3-11, GE Healthcare Inc.), 0.01% (w/v) bromophenol blue) to the final protein concentration of 1 mg/ml. IPG strips (7 cm, pH 3-11NL, GE Healthcare Inc.) were saturated overnight with 120 µl of this solution and subjected to isoelectric focusing in a GE Healthcare Ettan IPGphor 3 Isoelectric Focusing Unit (3 hours, total voltage of 7000 V). Subsequently, IPG strips were equilibrated for 20 minutes in reducing buffer (6 M urea, 50 mM Tris, 30% (v/v) glycerol, 2% (w/v) SDS, 2% (w/v) DTT) followed by 20 minutes in alkylating buffer (6 M urea, 50 mM Tris, 30% (v/v) glycerol, 2% (w/v) SDS, 2,5% (w/v) iodoacetamide). The second dimension was resolved on 12% 1 mm-thick SDS-PAGE mini gels, with the separation voltage of 130 V. For each experiment, a set of three or four identical gels was prepared. One gel was stained with Page Blue Coomassie staining solution (Fermentas Inc.) according to the manufacturer’s instructions. The other gels were prepared for the further immunoblotting.