4.4. Protein Isolation, 1-D SDS-PAGE, and Immunoblot Analysis

Total proteins were extracted from 100 mg of frozen nodules with 1 mL of SDS sample buffer (2% SDS, 60 mM Tris-HCl, pH 6.8, 5% β-mecaptoethanol). The samples were boiled at 100 °C for 5 min followed by centrifugation at 15800× g for 5 min. The resulting supernatant was treated as the total nodule protein fraction and was resolved by 15% SDS-PAGE gels using a Hoeffer SE 250 mini-vertical electrophoresis apparatus (GE Healthcare, Pittsburgh, PA, USA). Resolved proteins were visualized by staining overnight with Coomassie Brilliant Blue. Western blot analysis was performed as described earlier [75]. Polyclonal antibodies raised against leghemoglobin, malate dehydrogenase, AspAT-1, and AspAT-2 were obtained from Dr. Carol Vance (USDA-ARS). These antibodies were used at 1:5000 dilution. Sesbania nodule proteins specifically reacting against individual antibodies were detected with the aid of an enhanced chemiluminescent substrate (SuperSignal West Pico kit, Pierce, Rockford, Il, USA).