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Validation of hordein extraction methods.

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posted on 01.03.2013, 10:55 by Gregory J. Tanner, Malcolm J. Blundell, Michelle L. Colgrave, Crispin A. Howitt

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.

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