Immunoprecipitation and Western Blot Analysis of AP-1 Clathrin-Coated Vesicles

Abstract

The function and integrity of epithelial cells depends on the polarized localization of transmembrane proteins at either apical or basolateral plasma membrane domains. To facilitate sorting to the basolateral domain, columnar epithelial cells express the tissue-specific AP-1B complex in addition to the ubiquitously expressed AP-1A. Both AP-1A and AP-1B are heterotetrameric clathrin adaptor protein complexes that are closely related. Here we describe a biochemical method to separate AP-1B from AP-1A clathrin-coated vesicles by immunoprecipitation from clathrin-coated vesicle pellets that were obtained by ultracentrifugation and analyzed by SDS-PAGE and western blot using fluorescently labeled secondary antibodies.

Keywords: AP-1; Cell polarity; Clathrin-coated vesicles; Epithelial cells; Immunoprecipitation; Recycling endosomes; Trans-Golgi network.