The plasma membranes of polarized epithelial cells are divided into two distinct domains separated by tight junctions and characterized by distinct polypeptide compositions. Study of the cellular mechanisms involved in generating this anisotropy has recently been facilitated through the introduction of a surface labeling technique employing NHS-biotin (29). Incorporation of the biotin marker allows proteins present at one or the other cell surface domain to be identified and isolated. This methodology is applicable to investigations of both the steady state and the dynamic aspects of epithelial polarity. We discuss protocols through which this technique can be applied to various experimental situations. Methodologic details and special considerations are discussed. The utility of surface biotinylation for studies of epithelial polarity depends on this technique's capacity to report quantitatively on the surface distributions of plasmalemmal proteins. We find that under certain circumstances, surface biotinylation is extremely inefficient. Furthermore, in some cases the efficiency of surface biotinylation is dramatically different at the two plasmalemmal domains of epithelial cells grown on permeable filter supports. Thus, surface biotinylation does not always provide an accurate picture of plasma membrane protein distributions. These situations are discussed and methods to assess the efficiency and accuracy of surface biotinylation are proposed.
ASJC Scopus subject areas
- Pathology and Forensic Medicine