Abstract
When properly solubilized with trifluoroacetic acid (TFA), α- and β-tubulin subunits from a variety of sources may be resolved at high yield by reversed-phase high-performance liquid chromatography (HPLC), using a Waters μBondapak C 18 column and simple linear aqueous acetonitrile gradients containing TFA. The tubulin subunits are typically the most non-polar proteins present, with the β-tubulin subunit eluting before the α. Column temperatures above ambient improve both the resolution and the yield; less polar solvent systems do not. Tubulins not freely soluble in aqueous TFA may be solubilized in 6 M guanidine-hydrochloric acid with no change in retention time. Other columns with shorter carbon chain lengths and larger pore size produce a single, unresolved tubulin peak. Reversed-phase HPLC analysis provides an independent comparative evaluation of organelle-specific tubulins, with characteristic retention time differences observed between homologous ciliary and flagellar outer doublet tubulin subunits and also between them and their cytoplasmic counterparts.
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