Purification of mammalian tubulins and tubulin-associated proteins using a P2A-based expression system

Abstract

The microtubule cytoskeleton plays a crucial role in a myriad of cellular events, including mitosis, cell differentiation, migration, and the maintenance of cell shape. Microtubules are assembled from α- and β-tubulin heterodimers, whose biosynthesis is a complex process requiring the balanced production of α- and β-tubulin subunits. This chapter focuses on a method for the combined expression of tagged α- and β-tubulin dimers, their purification, and the isolation of co-purifying tubulin-associated proteins (TAPs) in mammalian cells. This approach is currently used in our laboratory to study tubulin function and to identify and characterize TAPs.