Xcell mini cell system

The polypeptide banding profiles of duplicate canola meal samples were visualised utilising an adapted SDS-PAGE procedure [40 (link)] as follows; the sample (~10 mg CP) was dissolved in sample buffer (1 mL: 11.25 mM tris-HCl, pH 8.5, 3.6% SDS, 18% glycerol, and 0.0025% bromophenol blue), and heated at 85 °C for 10 min. For reducing conditions, 50 mM dithiothreitol (DTT) was added to the sample buffer. The protein sample (30 µg of CP per well) and standard marker (5 µL, Novex Mark 12, Invitrogen, Mulgrave, VIC, Australia) were loaded onto a NuPAGE gradient precast gel (4–12% gradient) bis-tris (10 × 10 cm²) in a Novex Xcell mini cell system (Invitrogen, Mulgrave, VIC, Australia). Electrophoresis was performed at 80 V for 75 min, followed by 90 V for 75 min in running buffer (50 mM methysulfonic acid, 50 mM tris base, 0.1% SDS, 1 mM ethylenediaminetetraacetic acid, pH 7.3). The polypeptide bands were visualised by incubating the gel in Coomassie Brilliant Blue R-250 solution (0.1% in 40% methanol, 10% acetic acid) for 25 min, and de-stained (10% ethanol and 7.5% acetic acid) on an orbital shaker at RT overnight.

The native protein profiles of duplicate canola meal samples were visualised by gel-electrophoresis following manufacturer’s instructions. In brief, sample (~10 µg CP per µL) was added to 2.5 µL NativePAGE^TM sample buffer (4×), 1 µL NativePAGE^TM 5% G-250 sample additive, and made to 10 µL with deionised H₂O. The sample and NativeMark^TM unstained protein standard (5 µL, LC0725, Invitrogen, Mulgrave, VIC, Australia) were loaded onto a NativePAGE^TM 4–16% gradient precast bis-tris (10 × 10 cm²) gel in a Novex Xcell mini cell system (Invitrogen, Mulgrave, VIC, Australia). The running buffer contained 50 mM BisTris, 50 mM tricine, pH 6.8, and the sample buffer contained 50 mM BisTris, 6 N HCl, 50 mM NaCl, 10% w/v glycerol, and 0.001% Ponceau S, pH 7.2. The upper (inner) buffer chamber contained cathode buffer (200 mL: 10 mL NativePAGE^TM running buffer 20×, 10 mL NativePAGE^TM cathode additive 20×) and the lower (outer) buffer chamber contained anode buffer (600 mL: 50 mL NativePAGE^TM running buffer 20×, 950 mL deionised H₂O). Electrophoresis was performed at 150 V for 110 min. The native proteins were visualised by incubating the gel in 40% methanol and 10% acetic acid for 25 min, Coomassie Brilliant Blue R-250 solution (0.02% in 30% methanol and 10% acetic acid) for 25 min, and 8% acetic acid on an orbital shaker at RT overnight.