Gliadins and glutenins were extracted from wheat flour using a modified classical Osborne procedure based on protein solubility [16] .
The gliadin fraction from 100 mg of flour was extracted stepwise three times with a 670 µl of 60% (v/v) ethanol, vortexing for 2 min at room temperature (RT) and continued with incubation at RT 10 min with shaking. Samples were centrifuged at 6,000 x g. for 20 min, supernatants were collected and mixed all together. Glutenin fraction was extracted from the insoluble pellet stepwise two times with 500 µl of 50% (v/v) 1-propanol, 2 M urea, 0.05 M Tris-HCl (pH 7.5) and 2% (w/v) DTT, vortexing for 2 min at RT and incubation for 15 min at 60°C with shaking. Samples were centrifuged at 6,000 x g. for 20 min, supernatants were collected, mixed all together and filtered through a 0.45 µm nylon filter (Teknokroma). Gliadin (40 µl) and glutenin (40 µl) extracts were applied to a 300SB-C8 reverse phase analytical column (4.6×250 mm, 5 µm particle size, 300 Å pore size; Agilent Technologies) using a 1200 Series Quaternary LC System liquid chromatograph (Agilent Technologies) with a DAD UV-V detector, as described in [13] , [16] . Quantitative determination of gluten protein types in wheat flour was carried out by RP-HPLC. Absorbance was monitored with the DAD UV-V module at 210 nm. The integration procedure was handled automatically by the software with some minor manual adjustment. Absolute amounts of gliadin and glutenin fractions were determined using bovine serum albumin (BSA; BSA ≥98%, fraction V. Sigma-Aldrich, St Louis, MO, cat. no. A3294) as protein standard. Three independent repetitions were carried out for each transgenic line and control.
The gliadin fraction from 100 mg of flour was extracted stepwise three times with a 670 µl of 60% (v/v) ethanol, vortexing for 2 min at room temperature (RT) and continued with incubation at RT 10 min with shaking. Samples were centrifuged at 6,000 x g. for 20 min, supernatants were collected and mixed all together. Glutenin fraction was extracted from the insoluble pellet stepwise two times with 500 µl of 50% (v/v) 1-propanol, 2 M urea, 0.05 M Tris-HCl (pH 7.5) and 2% (w/v) DTT, vortexing for 2 min at RT and incubation for 15 min at 60°C with shaking. Samples were centrifuged at 6,000 x g. for 20 min, supernatants were collected, mixed all together and filtered through a 0.45 µm nylon filter (Teknokroma). Gliadin (40 µl) and glutenin (40 µl) extracts were applied to a 300SB-C8 reverse phase analytical column (4.6×250 mm, 5 µm particle size, 300 Å pore size; Agilent Technologies) using a 1200 Series Quaternary LC System liquid chromatograph (Agilent Technologies) with a DAD UV-V detector, as described in [13] , [16] . Quantitative determination of gluten protein types in wheat flour was carried out by RP-HPLC. Absorbance was monitored with the DAD UV-V module at 210 nm. The integration procedure was handled automatically by the software with some minor manual adjustment. Absolute amounts of gliadin and glutenin fractions were determined using bovine serum albumin (BSA; BSA ≥98%, fraction V. Sigma-Aldrich, St Louis, MO, cat. no. A3294) as protein standard. Three independent repetitions were carried out for each transgenic line and control.
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