Hordeins do not bleed off in aqueous washes.

<p>Duplicate 50 mg aliquots of Sloop flour were extracted in 1 mL of 50% IPA containing 1% (w/v) DTT (IPA/DTT) and an aliquot containing 20 µg protein dried in a SpeedyVac, dissolved in Urea/SDS and resolved on duplicate SDS-PAGE gels (lanes P) which were either stained in colloidal Coomassie Blue (A and C) or blotted to nitrocellulose (iBLOT Promega), blocked in PBST containing 5% skim milk powder, 1% (w/v) Tween overnight at 4°C. The blot was exposed to anti-gliadin-HRP (Sigma) diluted at 1/2000 for 30 min, then washed in PBST, and the signal produced by ECL reagent (Amersham) exposed to Hyperfilm (Amersham) (B and D). Similarly, 50 mg of duplicate flour samples were first washed in 1 mL of water, and centrifuged to give a supernatant and a pellet, which was dissolved in IPA/DTT. Aliquots containing 20 µg protein from both pellet (P1) and supernatant (S1) were dried, redissolved and subject to SDS-PAGE as above. The process was repeated with additional aqueous washing steps to produce supernatants S2–4 and pellets P2–4. Low molecular weight proteins were removed from pellets (A: arrowed) and extracted into supernatants (C: arrowed) after 2 washes. Proteins were calibrated with Benchmark protein ladder (St; Invitrogen).