β1 Integrin Cell-surface Immunoprecipitation (Selective Immunoprecipitation)

Abstract

Immunoprecipitation (IP) is a widely used method to isolate a specific protein from a mixed protein sample using an antibody that exclusively binds to that particular protein. This technique allows studying protein-protein and protein-nucleic acid interactions or to identify post-translational protein modifications. Many proteins, in particular cell surface receptors, localize to different compartments within cells where they elicit distinct functions by interacting with specific proteins. Integrins represent a major family of cell surface receptors consisting of non-covalently associated α and β subunits that mediate the interaction of cells with their environment. However, integrins do not only localize to the cell surface but are also present in other compartments including the endoplasmic reticulum and endosomes where they engage with a distinct set of interacting partners or show distinct post-translational modifications. Standard immunoprecipitation of β1 integrins from a cell lysate without prior fractionation isolates β1 integrins from all compartments. In contrast, selective immunoprecipitation of cell surface β1 integrin allows enriching for the pool of β1 integrin on the cell surface thereby minimizing contaminations with β1 integrins from other subcellular compartments. To achieve this, living cells are incubated with a β1 integrin-specific antibody on ice to label cell surface β1 integrins prior to cell lysis and precipitation.