Purification of Cytoskeletal Proteins by Fast Protein Liquid Chromatography (FPLC) Using an ÄKTA Start System.

Actin, myosin, and tubulin are ubiquitous components of the fibrous network known as the cytoskeleton. Cytoskeletal proteins are involved in a plethora of intracellular processes such as maintenance of cellular organization, organelle translocation, and various nuclear roles including chromosome separation during mitosis. Early methods for protein extraction primarily relied on the salting-out method which was performed in conjunction with biochemical assays. Since the advent of recombinant molecular biology, protein tagging has been coupled with chromatography to obtain highly purified proteins required for sensitive assays. This chapter provides a general standard operating procedure (SOP) for using the ÄKTA™ Start System controlled by UNICORN software for fast protein liquid chromatography (FPLC) of 6× his-tagged cytoskeletal proteins. The protocol can readily be modified for affinity and non-affinity purification techniques using the various ÄKTA™ Chromatography Systems.