Validation of hordein extraction methods.
Half seed extracts were sequentially extracted from cv Sloop (A), Risø 56 (B), Risø 1508 (C), ULG 2.0 (D) in water (1), then 0.5 M NaCl (2), then IPA/DTT (3), then Urea/DTT (4), and 20 µg of total protein from each extract was subject to SDS-PAGE, blotted, interrogated with commercial anti-gliadin-HRP (Sigma) and compared to standard proteins (M; Benchmark Protein Ladder, Invitrogen). Note lanes 2 and 3 are reversed in panels B and C. This blot was deliberately overloaded with protein, to maximise detection of hordeins in the aqueous and salt fractions, causing over exposure of lane 3. When loaded at a lower protein loading of 2 µg per lane a well resolved banding pattern was seen as in Fig. 1A.