Generation of chicken polyclonal antibodies against distinct maize isotubulins

Antibodies specific to five different maize isotubulins were made. From pre dicted amino acid sequences established from previously sequenced maize tub ulin genes, peptide antigens were synthesized matching the carboxyl-termina l 11-13 amino acids of each of three maize a-tubulins and two maize beta-tu bulins. Antibodies were generated by Injecting conjugated antigens into hen s, collecting their eggs, and extracting immunoglobulin Y from the egg yolk . Specificity of each antibody was tested by immunoblotting of fusion prote ins containing the antigenic sequence of the specific alpha- and beta-tubul in isoforms. For all five isotubulins, antibodies were affinity-purified wi th fusion proteins corresponding to their respective antigens, to remove no nspecific binding found in the antibody preparations. Further preparation o f anti-alpha-tubulins was required to eliminate cross-reactivity of antibod ies with members of other alpha-tubulin subfamilies. For this, affinity-pur ified antibodies against a specific alpha-tubulin were preadsorbed with pep tides representing cross-reactive alpha-tubulin antigens. Results indicated that virtually all crossreactivity between members of different alpha-tubu lin subfamilies could be eliminated, resulting in labeling of only the fusi on protein containing the specific antigen. All five isotubulin antibodies generated showed labeling of discrete spots on two-dimensional immunoblots of maize proteins, demonstrating the specificity of the antibodies in compl ex tubulin mixtures. These antibodies should prove valuable for analyzing t he developmental distribution, and possible functional significance, of sev eral maize isotubulins.