Two-Dimensional Gel Electrophoresis Workflow

For the first dimension of separation, nine dry strips of Immobiline strips (measuring 24 cm and at a pH of 3–11; GE Healthcare, Danderyd, Sweden) were passively rehydrated (30 V; 12 h), followed by isoelectric focusing using an Ettan IPGphor IEF unit (GE Healthcare, Danderyd, Sweden). The focusing was performed at 20 °C, with 50 µA per strip. After the first dimension, the strips were equilibrated and separated on 12.5% SDS-PAGE gels using an Ettan Dalt Six device (GE Healthcare, Danderyd, Sweden). The gels were then scanned using a Sapphire Biomolecular Imager (Azure Bio systems, Dublin, OH, USA), and digitalization was performed using the image analysis software Sapphire Capture system (Azure Biosystems, Dublin, OH, USA). Preparative gels were prepared using the total protein (1 mg) obtained from a pool of equal protein amounts. The gels were then stained using Colloidal Coomassie Blue, as described previously [17 (link),30 (link),31 (link)].