39] Purification and assay of microtubule-associated proteins (MAPs)

Abstract

Publisher Summary This chapter presents procedure for purification and assay of microtubule-associated proteins (MAPS). The procedures begin with microtubules assembled in vitro from either brain or tissue culture cells. The solution of microtubule proteins is usually carried through at least two to three complete assembly-disassembly cycles prior to beginning one of the following MAP isolation procedures. The strategies employed to isolate MAPs from the total microtubule protein preparation are based on several criteria: (1) net charge distribution affecting their behavior on ion exchange resins, (2) overall molecular size and axial ratio, which affect their elution from molecular sieve columns, and (3) varying stability to the effects of elevated temperatures (100°). One or more combinations of these properties can be used as the basis for the isolation procedures. Both the high molecular weight MAPs and Tau from brain and the MAPs from tissue culture cells have been assayed by their ability to promote the assembly of tubulin in vitro. Generally, these assays are carried out by determining whether the putative MAP will enhance the initial rate and total amount of assembly of a given concentration of PC or DEAE-purified brain tubulin.