Tubulin isotype specificity and identification of the epitope for antibody Tub 2.1

Tub 2.1, a monoclonal antibody commonly used to measure cellular tubulin content, is widely believed to recognize all beta-tubulin isotypes. Though it has been used for more than two decades, the epitope for this antibody is not well established. We report for the first time that contrary to common belief, this antibody does not react with all isotypes of beta-tubulin. Of the seven vertebrate beta-tubulins, the more divergent class V and VI isotypes are not recognized by this antibody. Among the isotypes that do react, binding is similar for beta2, beta3, beta4a and beta4b but lower for beta1, the most abundant isotype. Expression of chimeric tubulins verified that the epitope for Tub 2.1 is near the C-terminal end of beta-tubulin. Site-directed mutagenesis of this region in nonreactive beta5 to match the sequence of beta4b resulted in strong reaction to Tub 2.1 and narrowed the epitope to amino acids 431-436.