This chapter presents procedure for preparation of tubulin from brain. Two methods for preparing tubulin is presented (a) purification by cycles of assembly and disassembly followed by chromatography on phospbocellulose (b) purification by the modified Weisenberg procedure, each of which yields several hundred milligrams of purified protein. The principal properties of the tubulin prepared by the two methods appear to be identical and a choice of one route or the other can be made on the basis of available apparatus or of the investigator's familiarity with the manipulations involved. The tubulin that results from either preparation is substantially free of microtubule-associated proteins. The protein concentration of the solution is determined spectrophotometrically in 6 M guanidine hydrochloride at 275 nm with an absorptivity value of 1.03 ml/(mg cm). It is then adjusted to 50-60 mg/ml and rapidly frozen in small aliquots to be stored in liquid nitrogen. Under these conditions the protein can be stored for at least 14 days without observable loss of colchicine binding activity or of its ability to undergo in vitro microtubule assembly.
ASJC Scopus subject areas
- Molecular Biology