Chapter 1 - Preparation of Microtubule Protein and Purified Tubulin from Bovine Brain by Cycles of Assembly and Disassembly and Phosphocellulose Chromatography

Abstract

This chapter presents an efficient, high yield, and relatively easy protocol for purification of microtubule protein (MTP) (tubulin plus stabilizing microtubule-associated proteins (MAPs), consisting of ∼70–75% tubulin and 25–30% MAPs), and subsequently for purifying tubulin from the MTP in the absence of assembly-promoting solvents. The protocol for purification of MTP (tubulin plus MAP) in the absence of glycerol begins with two steer brains, weighing a total of between 400 and 600 g. Processing this quantity of tissue requires the equivalent of six Sorvall refrigerated superspeed centrifuges. Brains are initially blended in a Waring Commercial Blender at a ratio of 1.5 ml of buffer per gram of wet brain weight at low speed for 30 s. Then, the blended brains are homogenized by using one pass in a motor-driven Teflon pestle/glass homogenizer operated at the maximum speed. The MTP, processed through three cycles of warm assembly and cold disassembly, is often used in this protocol. This third cycle involves centrifuging through 50% sucrose cushions to remove any proteins that do not adhere to the microtubules. An alternative to the third cycle for purification of MTP (tubulin plus MAPs) is to purify tubulin devoid of MAPs beginning with the C2S using phosphocellulose column chromatography.