PREPARATION OF ANTI-MUCIN POLYPEPTIDE ANTISERA TO STUDY MUCIN BIOSYNTHESIS

Mucins are very heavily O-glycosylated glycoproteins. For in depth stu dies on the cell biological aspects of mucins, anti-polypeptide antibo dies are essential. We therefore developed a method for the preparatio n and screening of polyclonal antisera against mucin peptide epitopes, Mucins from five different tissues were isolated using CsCl/guanidini um.HCl density gradient centrifugation, and polyclonal antisera were p repared. Specificity for mucin peptide epitopes was determined by West ern blotting, immunohistochemistry, and immunoprecipitation. The versa tility of each anti-mucin antiserum for the study of mucin biosynthesi s was tested in metabolic labeling experiments on tissue explants. All polyclonal antisera were directed primarily against peptide epitopes of mucin precursors as well as of fully glycosylated mucins. Each of t he polyclonal antisera enabled us to study the mucin biosynthesis in t he organ where the mucin was isolated from originally. Our mucin isola tion method yields very pure mucins with sufficiently intact polypepti des to reproducibly elicit polyclonal anti-polypeptide antisera. As th e sera recognized the polypeptides, primarily independent of the state of O-glycosylation, the intermediate steps in the biosynthesis of the mucins could be identified. (C) 1995 Academic Press, Inc.