Separation of tubulin subunits by reversed-phase high-performance liquid chromatography.
When properly solubilized with trifluoroacetic acid (TFA), alpha- and beta-tubulin subunits from a variety of sources may be resolved at high yield by reversed-phase high-performance liquid chromatography (HPLC), using a Waters muBondapak C18 column and simple linear aqueous acetonitrile gradients containing TFA. The tubulin subunits are typically the most non-polar proteins present, with the beta-tubulin subunit eluting before the alpha. Column temperature 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.
Stephens
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Marine Biological Laboratory, Woods Hole, MA 02543.
D000818:Animals; D002851:Chromatography, High Pressure Liquid; D002923:Cilia / Q000032:analysis; D003593:Cytoplasm / Q000032:analysis; D004591:Electrophoresis, Polyacrylamide Gel; D005407:Flagella / Q000032:analysis; D006146:Guanidines; D006454:Hemoglobins / Q000302:isolation & purification; D010084:Oxidation-Reduction; D011506:Proteins / Q000302:isolation & purification; D012617:Sea Urchins / Q000378:metabolism*; D012997:Solvents; D013056:Spectrophotometry, Ultraviolet; D013696:Temperature; D014404:Tubulin / Q000302:isolation & purification*
D006146:Guanidines; D006454:Hemoglobins; D011506:Proteins; D012997:Solvents; D014404:Tubulin