IDENTIFICATION OF GLYCOSYLATED FORMS OF WHEAT STORAGE PROTEINS USING 2-DIMENSIONAL ELECTROPHORESIS AND BLOTTING

Two-dimensional electrophoresis with acid-polyacrylamide gel electroph oresis (PAGE), followed by sodium dodecyl sulfate (SDS)-PAGE and SDS-P AGE of unreduced polypeptides followed by SDS-PAGE under reducing cond itions, were used to separate and identify the different subgroups of gliadins and glutenins and to distinguish between covalent and noncova lent polymers of glutenins. Gels were blotted under semidry conditions according to Lauriere (Anal. Biochem. 1993, 212, 206-211) to allow la rge polymers of glutenins to be transferred efficiently. Glycosylated polypeptides were detected on blots using either the method of Haselbe ck and Hosel (Glycoconjugate J. 1990, 7, 63-74), or using anti-(xylose -containing N-glycan) antibodies (Lauriere et al., Plant Physiol 1989, 90, 1182-1188). High and low molecular weight glutenin subunits were shown to aggregate through both disulfide bridges and noncovalent prot ein-to-protein interactions. Aggregated gamma-gliadins were also demon strated. Glycans were detected on both gliadin and glutenin polypeptid es. Covalently aggregated low molecular weight glutenins were shown to contain N-glycans with xylose, which demonstrated their sorting in th e Golgi apparatus.