Preparation of Myristoylated Arf1 and Arf6

Arf proteins are members of the Arf family of small Ras‐like GTP binding proteins. Six Arfs, grouped into three classes, have been identified in mammalian cells and three members have been identified in yeasts. Arf1 and Arf6, more extensively studied than other Arfs, have been found to affect membrane traffic and actin remodeling. A structural feature that distinguishes Arfs from other Ras superfamily members is an N‐terminal α‐helix, extending from the basic G‐protein fold, which is cotranslationally myristoylated. Both the helix and the myristate affect biochemical properties of Arfs, including nucleotide exchange, membrane association, and interaction with some effector proteins. Preparation of myristoylated Arf for in vitro studies of Arf function requires consideration of both the reaction yielding myristoylated protein and the properties of the modified Arfs. Here, we describe methods that yield homogeneous preparations of myristoylated Arf1 and Arf6.