The combination of Indian ink staining with immunochemiluminescence detection allows precise identification of antigens on blots: application to the study of glycosylated barley storage proteins.

The localization after blotting of specific spots in two-dimensional electrophoretic protein pattern was achieved using, in that order, Indian ink protein staining and immunodetection with chemiluminescence on the same membrane. Indian ink did not inhibit significantly the antibody reactions even after overnight staining. It produces permanent staining that did not quench the chemiluminescent signal, recorded on a film. This allowed perfect matching between the specific and the total protein patterns. The procedure was applied to the identification of glycoproteins present in barley storage protein preparations.

Identification of glycosylated forms of wheat storage proteins using two-dimensional electrophoresis and blotting.

Two-dimensional electrophoresis with acid-polyacrylamide gel electrophoresis (PAGE), followed by sodium dodecyl sulfate (SDS)-PAGE and SDS-PAGE of unreduced polypeptides followed by SDS-PAGE under reducing conditions, were used to separate and identify the different subgroups of gliadins and glutenins and to distinguish between covalent and noncovalent polymers of glutenins. Gels were blotted under semidry conditions according to Laurière (Anal. Biochem. 1993, 212, 206-211) to allow large polymers of glutenins to be transferred efficiently. Glycosylated polypeptides were detected on blots using either the method of Haselbeck and Hösel (Glycoconjugate J. 1990, 7, 63-74), or using anti-(xylose-containing N-glycan) antibodies (Laurière 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 protein-to-protein interactions. Aggregated gamma-gliadins were also demonstrated. Glycans were detected on both gliadin and glutenin polypeptides. Covalently aggregated low molecular weight glutenins were shown to contain N-glycans with xylose, which demonstrated their sorting in the Golgi apparatus.