Use of Antibodies for Studying the Sidedness of Membrane Components

Antibody molecules cannot diffuse across biological membranes. For this reason, an externally added antibody will only form an immune complex with a membrane component if the component is at least partly situated on a membrane surface facing the suspending medium. With closed membrane structures, such as intact cells or mitochondria, only components on the outer side of the limiting membrane will be able to react with added antibody. If the closed membrane system is inverted, components formerly situated on the inaccessible inner membrane face become accessible to antibody. Antibodies against individual membrane components are, thus, useful as specific tools for investigating the location of membrane components. This chapter reviews three major methods of utilizing specific antibodies for such studies: (1) inhibition of enzymatic activities, (2) binding of radioactively labeled immunoglobulins to membranes, (3) electron microscopic examinations of membrane vesicles that had been reacted with ferritin conjugated immuno-γ-globulins. With each of these three methods, it should be ascertained that incubation with antibodies does not damage or invert the membranes under study. As with other methods for probing membrane sidedness, this is frequently the most difficult part of the investigation.