Imagemaster 2d software

Before electrophoresis, 200 μg of total PM proteins was mixed in 350 mL of rehydration buffer I (8.0 M urea, 2% (v/v) CHAPS, 0.8% (w/v) DTT, 0.5% IPG buffer, pH 3–10, 0.002% (w/v) bromophenol blue) and loaded onto an 18 cm broad range IPG strip (pH 3 to 10 NL; GE Healthcare, Milwaukee, MI, USA). Isoelectric focusing (IEF) was done at 20 °C according to the following protocol: 50 V for 12 h, 100 V for 1 h, 200 V for 1 h, 500 V for 30 min, 1000 V for 30 min, 3000 V for 30 min, 5000 V for 30 min then 8000 V until 100,000 Vh. The current limit is 50 μA per IPG strip. Before SDS-PAGE, the IPG strips were equilibrated for 15 min in equilibration buffer I (6 M urea, 50 mM Tris-HCl, pH 8.8, 2% (w/v) SDS, 30% (v/v) glycerol, 1% (w/v) DTT) and later for a further 15 min in equilibration buffer II (equilibration buffer I containing 2.5% (w/v) iodoacetamide instead of DTT). Equilibrated strips were overlaid onto 15% (w/v) polyacrylamide gel and subjected to 2-DE in an Ettan DALTsix Electrophoresis System (GE Healthcare, Milwaukee, MI, USA) followed by staining with silver nitrate [39 (link)]. At least three biological replicates were performed for PM protein samples. Spots were scanned with a high-resolution image scanner III at 300 pixels and analyzed by ImageMaster 2D software (GE healthcare, Milwaukee, MI, USA).