4.3. Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE)

SDS-PAGE analysis was performed to assess protein composition and intensity following a previously published protocol [55]. Total glutens were fractionated by SDS-PAGE for HMW-GS profiling. After the glutenin supernatant (1 mL) was removed by drying, 100 µL of 2X sample buffer [1.8% SDS, 12% glycerol, 54 mM Tris-HCl (pH 8.8), 1.8 mM EDTA (pH 8.0), and 0.006% bromophenol blue (BPB)] was added. After incubation at 65 °C for 15 min, 50 µL of sample was run on a 12% SDS-PAGE gel (30% acrylamide, 1.5 M Tris-HCl (pH 8.8), and 10% SDS) at 200 V for 18 h in a running buffer composed of 0.192 M glycine, 0.1% SDS, and 0.025 M Tris base. HMW-GSs were visualized on 12% SDS-PAGE gels (12% (w/v) acrylamide) and stained with staining solution (10% glacial acetic acid, 50% methanol, and 0.1% Coomassie Brilliant Blue R-250 (CBB)). The same solution without CBB was used to destain the gels.