Multiphor 2 electrophoresis unit

2DE was performed for gluten separation by GENOMINE (Seoul, Korea). Briefly, IPG dry strips (4–10 NL IPG, 24 cm, Genomine, Korea) were equilibrated for 12–16 h with 7 M urea, 2 M thiourea containing 2% 3-[(3-cholamidopropy) dimethyammonio]-1-propanesulfonate (CHAPS), 1% dithiothreitol (DTT), and 1% Pharmalyte. IPG dry strips were loaded with 200 µg of gluten. Isoelectric focusing (IEF) was performed at 20 °C using a Multiphor II electrophoresis unit and an EPS 3500 XL power supply (Amersham Biosciences, Little Chalfont, UK) following the manufacturer’s instructions. For IEF, the voltage was increased linearly from 150 to 3500 V with focusing complete after 96 kVh. Before the second dimension, strips were incubated for 10 min in equilibration buffer (50 mM Tris-Cl, pH 6.8 containing 6 M urea, 2% SDS, and 30% glycerol) first with 1% DTT and then with 2.5% iodoacetamide. The equilibrated strips were then inserted onto SDS-PAGE gels (20 × 24 cm, 10–16%). SDS-PAGE was performed using a Hoefer DALT 2D system (Amersham Biosciences, Little Chalfont, UK). We ran the 2D gels at 20 °C and 1700 Vh. Separated protein fractions were silver-stained as described previously [56 (link)].

Immobilized pH gradient (IPG) strips (pH 3–10, 24 cm) were rehydrated and the prepared samples were applied with cup loading. Isoelectric focusing was performed with a Multiphor™ II electrophoresis unit (Amersham Biosciences, Little Chalfont, Bucks, UK) for 54 kVh at 20°C in the dark [19 , 20 (link)]. The strips were equilibrated for 10 min in buffer (50 mM Tris-HCl [pH 8.8], 6 M urea, 30% [v/v] glycerol, 1% [w/v] sodium dodecyl sulfate [SDS]) containing 65 mM dithiothreitol, and then for 10 min in the same buffer containing 240 mM iodoacetamide. The equilibrated IPG strips were transferred onto 24 cm × 20 cm, 12% T, 7.5% C polyacrylamide gels made between low-fluorescence glass plates. The strips were overlain with 0.5% (w/v) low-melting-point agarose in buffer (25 mM Tris-base, 0.1% SDS, 192 mM glycine) containing 0.1% bromophenol blue. The gels were run in the Ettan DALT Twelve Electrophoresis System (Amersham Biosciences) at 2 W/gel at 20°C, until the dye fronts had run off the bottom of the gels. The two-dimensional (2-D) gels between low-fluorescence glass plates were scanned directly with a Typhoon 9400 imager (Amersham Biosciences). Normalization of the three Cy™ dyes was accomplished by adjusting the maximum pixel values to 55.000 counts by changing the photomultiplier tube voltage. The images generated were exported as tagged images (Amersham Biosciences).

Immobilized pH gradient dry strips (4–10 NL immobilized pH gradient, 24 cm, Genomine, Korea) were equilibrated for 12–16 h with 7 M urea, 2 M thiourea containing 2% 3-([3-cholamidopropy] dimethyammonio)-1-propanesulfonate, 1% dithiothreitol, and 1% pharmalyte, and loaded with 200 μg of sample. Isoelectric focusing was performed at 20 °C using a Multiphor II electrophoresis unit and EPS 3500 XL power supply (Amersham Biosciences, UK) following the manufacturer’s instructions. For isoelectric focusing, the voltage was linearly increased from 150 to 3500 V over 3 h for sample entry followed by a constant 3500 V, with focusing complete after 96 kV/h. Prior to the second dimension, strips were incubated for 10 min in equilibration buffer (50 mM Tris-Cl, pH 6.8 containing 6 M urea, 2% sodium dodecyl sulfate [SDS], and 30% glycerol), first with 1% dithiothreitol and second with 2.5% iodoacetamide. Equilibrated strips were inserted onto SDS-PAGE gels (20 × 24 cm, 10–16%). SDS-PAGE was performed using the Hoefer DALT 2D system (Amersham Biosciences, UK) following the manufacturer’s instruction. Two dimensional gels were run at 20 °C for 1700 V/h and then the 2D gels were stained with colloidal Coomassie brilliant blue as described by Oakley et al. [33 (link)], although the fixing and sensitization step with glutaraldehyde was omitted.