Chapter 22 - Preparation of Tubulin from Porcine Brain

Abstract

This chapter describes the procedure for preparation of tubulin from porcine brain. Microtubules are polymers of the heterodimeric protein tubulin. Because these heterodimers have the same orientation in the microtubule lattice and the dimers themselves are not symmetric, it follows that the microtubule itself must have an inherent polarity. Since the principle of tubulin preparation relies on successive rounds of polymerization and depolymerization steps based on temperature shifts, delays and not having the right equipment at the right temperature will reduce the yield drastically. The BRB80 conversion buffer is added to the purified tubulin that has been eluted from the phosphocellulose column to convert the buffer from column buffer composition to BRB80 prior to storage of the tubulin. Nucleotides are made as stock solutions as 100 mM ATP and 200 mM GTP. It is important that the pH of these nucleotides does not become acidic, as hydrolysis will result. The brains are then homogenized twice for approximately 30 s each. Typically, it can be expected to need approximately 5 l of BB for 30 porcine brains giving about 10 l of total homogenate.