Two-Dimensional Gel Electrophoresis Proteomics

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).