Isolation of mammalian brain tubulin by amino-activated gel chromatography.

In the present study, we report the isolation of the acidic structural protein tubulin using a number of amino-activated gels. Crude 100,000 g supernatant derived from sheep brain was applied to gels activated with either aminohexyl, aminoethyl, argininyl, diethylaminoethyl, lysinyl and polylysinyl residues and eluted with three distinct sequential buffer changes (pH 6.5): (i) 0.025-0.4 M morpholinoethanesulphonic acid; (ii) 0.076 and 0.379 M ammonium sulphate in 0.025 M morpholinoethanesulphonic acid; and (iii) 0.8 M sodium chloride in 0.025 M morpholinoethanesulphonic acid. Tubulin was recovered from all columns in an enriched form. However, the elution profile and purity, as judged by [3H]colchicine binding and electrophoresis, varied with the ligand. Hydrophobic gels, such as diethylaminoethyl and aminohexyl, required elution with high-ionic-strength buffers (0.8 M sodium chloride) and significant inhibition of [3H]colchicine activity resulted. This problem was avoided with the hydrophilic ligands such as arginine, polylysine and aminoethyl. Manipulation of elution conditions enabled complete elution of tubulin from arginine-activated gels in 2.5% ammonium sulphate without detectable losses of [3H]colchicine binding activity and with purity comparable to that achieved using diethylaminoethyl Sephacel.