Eps 3500 xl power supply

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 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.