Monoclonal Antibodies as Context-Specific Reagents: Differential Antigen Reactivity of MAb 58K-9 During Immunoprecipitation,Immunoblotting,and Immunofluorescence Analyses

Abstract

Monoclonal antibodies (MAbs) are used as antigen-specific reagents in a variety of techniques. Usually it is assumed that antigen specificity shown by immunoprecipitation and immunoblotting will persist in immunofluorescence analyses. Here, we show that the behavior of MAbs is not always consistent with this assumption. Specifically, we demonstrate that a MAb (MAb 58K-9) preferentially interacts with the receptor-associated protein (RAP), a ubiquitous ER protein acting as a chaperone for the low density lipoprotein receptor-related protein (LRP) in immunoprecipitation and immunoblotting analyses. However, MAb 58K-9 does not recognize RAP in immunofluorescence studies. Interestingly, by this technique MAb 58K-9 exclusively detects an unrelated antigen peripherally associated with the cytosolic aspect of Golgi membranes. This reactivity was observed for primate but not rodent antigens. Our results indicate that MAbs that recognize a specific antigen by a single immuno-technique cannot be assumed to recognize the same antigen by other immuno-techniques, and that the identity of recognized antigens must be confirmed within each experimental context.