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.