Application of sequential extraction procedures and glycoprotein blotting for the characterization of the 2‐D polypeptide patterns of barley seed proteins

Abstract
Barley (Hordeum vulgare L.) proteins were sequentially extracted from ground seeds with Tris‐HCl buffer, 55% 2‐propanol, 55% 2‐propanol containing 1% dithiothreitol, and 6 M urea containing 2 % Nonidet P‐40 and 1 % dithiothreitol. The protein composition of these solubility fractions was then analyzed by high resolution two‐dimensional gel electrophoresis with immobilized pH gradient 4–9 in the first dimension, followed by silver staining and glycoprotein blotting, respectively, for a more detailed characterization of the two‐dimensional polypeptide pattern of barley seed proteins.