Staining of serum concanavalin a-binding proteins after micro two-dimensional electrophoresis and blotting

Abstract

Human serum proteins separated by micro two-dimensional electrophoresis in the absence of denaturants were electrophoretically transferred to nitrocellulose sheets (blots), and glycoproteins on the blots were stained by sequential incubation with the lectin concanavalin A, the glycoprotein horseradish peroxidase and the substrates of peroxidase diaminobenzidine-hydrogen peroxide. The stained serum concanavalin A-binding glycoproteins were identified by referring to the “identification map” of human plasma proteins, which we have prepared by immunochemical staining of the proteins on the blots. The concanavalin A-peroxidase method applied for the blots after two-dimensional electrophoresis provided information on the location of serum concanavalin A-binding glycoproteins, and showed greater sensitivity than Coomassie Brilliant blue R-250 staining for several serum proteins.