Ettan ipgphor manifold

For two-dimensional gel electrophoresis, the two samples to be run on the same gel plus the internal standard were mixed before adding 2 × GE lysis buffer (7 M urea, 2 M thiourea, 4% (w/v) CHAPS, 12 µ ml⁻¹ DeStreak reagent (GE Healthcare)) and 2% (v/v) ampholytes immobilized pH gradient (IPG) buffer (pH 3–10 NL, GE Healthcare) to a final volume of 125 µl. Isoelectric focusing (IEF) was carried out on pH 3–10 IPG-strips (24 cm, nonlinear gradient; GE Healthcare) using the IPGphor system from GE Healthcare. Immobiline DryStrips were rehydrated overnight with DeStreak rehydration solution (GE Healthcare) before cup-loading of proteins and IEF on an Ettan IPGphor Manifold (GE Healthcare). The migration was performed at 20°C (60 V for 2 h; gradient from 60 to 500 V for 5 h; hold 500 for 1 h, gradient from 500 to 1000 for 3 h; hold 1000 V for 1 h; gradient from 1000 to 8000 V for 4 h, hold 8000 V until 64 000 Vh). After the IEF, IPG strips were equilibrated twice for 15 min in equilibration buffer (50 mM Tris–HCl pH 8.8, 6 M urea, 30% (v/v) glycerol, 2% (w/v) SDS and 0.002% (w/v) bromophenol blue) supplemented with DTT and then with iodoacetamide. Second-dimension SDS–PAGE was performed using 24 cm format 12.5% resolving gel and run at 20°C overnight with 1.5 W per gel, using the Ettan DALT twelve system (GE Healthcare).

2D gel electrophoresis was performed for the analysis of proteins extracted from cholangiocarcinoma cells cultured in uncoated and 24-h matrix gel-coated plates. Each electrophoresis gel contained three pooled samples from the cell culture plates. Six gels were prepared in biological triplicates from the uncoated and matrix gel-coated plates. Protein samples (500 μg) were applied to 18-cm immobilized pH gradient (IPG) gel strips (pH 3–10; GE Healthcare, Uppsala, Sweden) by cup loading near the anodic ends of the strips. Isoelectric focusing (IEF) was performed using an Ettan IPGphor Manifold on an Ettan IPGphor isoelectric focusing unit (GE Healthcare) for 32,000 Vh at 20°C. Following IEF, each gel strip was equilibrated with equilibration buffer. The IPG strips were then loaded and run on 12.5% acrylamide gels (GE Healthcare) using the Ettan DALTsix electrophoresis system (GE Healthcare). The run was stopped after the bromophenol blue dye front had run off the bottom of the gels. The gels were then stained with colloidal Coomassie Blue (GE Healthcare).