Electrophoresis and LC-MS/MS analysis
One dimensional electrophoresis (1DE) : Meal and OB proteins were separated using SDS-PAGE in one dimension (1D) under both reducing and non-reducing conditions as described by Wanasundara et al. (2012). Purified cruciferin (1 mg/mL) and napin (4 mg/mL) were dissolved in 0.1 M phosphate buffer (pH 8.0) containing 0.1 M NaCl and also separated by 1DE. Homogeneous 20% polyacrylamide gels with 1.7-40 kDa molecular weight standards (SpectraTM Molecular Low-Range protein ladder, Thermo Fisher Scientific Inc.) were used for napin and cruciferin. 8-25% polyacrylamide gradient gels with 4.6–170 kDa molecular weight standards (PageRuler™ pre-stained protein ladder, Thermo Fisher Scientific Inc.) were used for all other proteins. Native-PAGE under non-denaturing conditions was performed for cruciferin and napin with reference proteins bovine serum albumin (66 kDa monomer and 132 kDa dimer) and jack bean urease (272 kDa trimer and 545 kDa hexamer (Sigma Aldrich Co.) as described by Perera et al. (2016).
Two-dimensional electrophoresis (2DE) : Purified proteins (cruciferin and napin at 1 mg protein/mL and OBPs at 1 μg/mL) were mixed with sample buffer (1:2, v:v) containing 6.7 M urea, 2% (w:v) 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonic acid (CHAPS), 0.5% carrier ampholytes, 0.001% bromophenol blue and deionized water. pH 3-10 ampholytes (Bio-Lyte® 3-10, Bio-Rad Laboratories Inc.) were used for cruciferin and OBPs, and pH 9-11 ampholytes (ZoomTM, Thermo Fisher Scientific Inc.) for napin. Immobilized pH Gradient (IPG) strips (pH 3-10 IPG for cruciferin and OBPs; pH 9-12 IPG for napin) were re-hydrated in each protein solution overnight at 4°C prior to isoelectric focusing (IEF). IEF was conducted using an IEF Cell (Bio-Rad Laboratories Inc.) for 2 h at 50 V, then 2 h at a voltage gradient from 200-4,000 V, and finally for 9 h at 4,000 V. Prior to running the second dimension, each protein strip was equilibrated for 15 min in buffer 1 [1.8 g urea, 1 mL of 10% SDS, 1.25 mL of 1.5 M Tris-HCl (pH 8.8), 1 mL of 100% glycerol and 0.6 mL of Milli Q water] followed by 15 min in buffer 2 [1.8 g urea, 1 mL of 10% SDS, 1.25 mL of 1.5 M Tris-HCl (pH 8.8), 1 mL of 100 % glycerol, 125 mg iodoacetamide and 0.6 mL of Milli Q water). The second-dimension separation (SDS-PAGE) was carried out using a Bio-Rad Mini-Protean® tetra cell (Bio-Rad Laboratories Inc.) with hand-cast 12% (for cruciferin) or 16 % polyacrylamide (for napin and OB protein) gels. The gels were stained with 0.1% Coomassie Blue R-250 and de-stained in 50% (v:v) methanol in water with 10% (v:v) acetic acid solution for 1-2 h to visualize separated proteins.