This chapter focuses on cell surface biotinylation and other techniques for determination of surface polarity of epithelial monolayers. The proteins present on the surface of the filter-grown monolayers can be selectively modified by the water soluble cell-impermeable biotin analog sulfo-NHSbiotin. Taking advantage of the access afforded by the filter support, the addition of sulfo-NHS-biotin to only one surface of the cell results in the selective labeling of only the apical or basolateral surface proteins. The amount of protein present on the apical or basolateral surface is determined by a densitometric analysis of the autoradiographs. Multiple exposures are necessary if using the film to insure that the values obtained are in the linear range of the film. Polarity is expressed as the percentage of total surface protein present on one surface of the monolayer. Some cell lines are better labeled from the apical surface. For apical pulse, starvation medium is removed from both chambers and pulse medium is applied only to the apical chamber.
ASJC Scopus subject areas
- Biochemistry, Genetics and Molecular Biology(all)