Generation of chicken polyclonal antibodies against distinct maize isotubulins

Antibodies specific to five different maize isotubulins were made. From predicted amino acid sequences established from previously sequenced maize tubulin genes, peptide antigens were synthesized matching the carboxyl-terminal 11-13 amino acids of each of three maize α-tubulins and two maize β-tubulins. Antibodies were generated by injecting conjugated antigens into hens, collecting their eggs, and extracting immunoglobulin Y from the egg yolk. Specificity of each antibody was tested by immunoblotting of fusion proteins containing the antigenic sequence of the specific α- and β-tubulin isoforms. For all five isotubulins, antibodies were affinity-purified with fusion proteins corresponding to their respective antigens, to remove nonspecific binding found in the antibody preparations. Further preparation of anti-α-tubulins was required to eliminate cross-reactivity of antibodies with members of other α-tubulin subfamilies. For this, affinity-purified antibodies against a specific α-tubulin were preadsorbed with peptides representing cross-reactive α-tubulin antigens. Results indicated that virtually all cross-reactivity between members of different α-tubulin subfamilies could be eliminated, resulting in labeling of only the fusion 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 complex tubulin mixtures. These antibodies should prove valuable for analyzing the developmental distribution, and possible functional significance, of several maize isotubulins.