NONRADIOACTIVE METHODS FOR LABELING AND IDENTIFYING MEMBRANE-SURFACE PROTEINS

The function of cilia is to provide motility, surfaces on which a variety of cell surface molecules are displayed for cell-cell interactions during mating, chemoreception, and a variety of cellular functions. In a study of Tetrahymena thermophila ciliary membrane proteins, identified were more than 40 ciliary membrane surface proteins, at least 16 of which also bind concanavalin A. Putative membrane surface proteins can be identified by iodination, followed by resolution of proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and autoradiography. Biotinylation involves the covalent coupling of biotin to a protein or other molecule and the subsequent binding of biotin to avidin or the more specific streptavidin, with which biotin forms an extremely high affinity bond. Avidin or streptavidin can be coupled to alkaline phosphatase or other reagents that produce a colorimetric reaction used to identify biotinylated proteins bound to nitrocellulose. A variety of compounds, some of which are reversible, can be used to link biotin to proteins, carbohydrates, and nucleic acids. Sulfosuccinimidyl 6-(biotinamido) hexanoate (NHS-LC biotin) reacts with primary amines (generallylysine epsilon groups) to form a stable covalent amide bond. Reactions are favored in slightly alkaline media, in which the primary amines are unprotonated. The NHS-LC biotin is water soluble and does not penetrate the ciliary membrane to label axonemal or cytoplasmic proteins. The method of identifying putative membrane proteins by staining blotted proteins with concanavalin A (Con A) and HRP has been described in this chapter. NHS-LC biotin has an extremely short half-life and virtually all labeling is completed within 5–10 minutes. These methods were developed for Tetrahymena cells, but similar methods have been used for Chlamydomonas. The chapter discusses the methods for labeling Tetrahymena cilia with NHS-LC biotin, alkaline phosphatase-streptavidin staining of biotinylated protein, labeling blotted proteins with concanavalin A. © 1995 Academic Press Inc.