Ettan daltsix vertical system

High-resolution 2-DE was performed according to Görg et al. [43 (link)] with some modifications as previously described by Franco et al. [41 (link)]. The first dimension was run in 24 cm long pH 4–7 linear gradient ReadyStrip^TM IPG Strips (Bio-Rad Laboratories, Hercules, CA, USA), loaded with 450 µg of protein from each sample, concurrently with 0.6% DTT and 1% immobilized pH gradient (IPG) buffer (Bio-Rad Laboratories). Isoelectric focusing (IEF) was performed on PROTEAN IEF Cell (Bio-Rad Laboratories). Initially, 50 V were applied for 12 h to rehydrate each strip, following a voltage grade until 70 kWh. Subsequently, strips were immersed in equilibration buffer I (50 mM Tris pH 8.8, 30% glycerol, 2% SDS, 6 M urea and 1% DTT) for 15 min at room temperature and in the same conditions with equilibration buffer II (50 mM Tris pH 8.8, 30% glycerol, 2% SDS, 6 M urea and 2.5% iodoacetamide). For the second dimension, proteins were resolved on 13% SDS-PAGE gels of 24 × 20 cm using Ettan DALTsix vertical system (GE Healthcare).

One-dimensional analytical gel electrophoresis was performed, followed by two-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) on 12.5% fixed concentration gels, as previously described [20 (link),21 (link),22 (link)]. Briefly, 1 mg from the pool was added to the rehydration buffer (7 M urea, 2 M thiourea, 4% CHAPS, 0.006 g DTT, 2 µL bromophenol blue, 5 µL IPG buffer (pI 3–11), a protease inhibitor cocktail) and applied to IPG strips. Isoelectric focusing was performed on an Ettan IPGphor IEF Unit (GE Healthcare, 30 V, 12 h, 20 °C at 50 µA per strip). Furthermore, the strips were treated by equilibration buffer with 0.1 g DDT and 0.9 g Iodoacetamide, respectively. Next, standard SDS-PAGE was preformed using an Ettan DALTsix Vertical System (GE Healthcare). The gels were scanned with a Sapphire biomolecular imager (Azure Biosystems, Dublin, OH, USA) and digitalized with Sapphire capture system image analysis software (Azure Biosystems, Dublin, OH, USA).