Establishment of a Sensitized Immunoblotting Method for Measuring Plant Tubulin Content

概要

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A sensitized immunoblotting method was established for measuring small amounts of plant tubulin. The method involves electrophoretic transfer of protein including tubulin from SDS-polyacrylamide gels onto nitrocellulose paper, successive incubation of the nitrocellulose paper with a mouse monoclonal antibody to α- or β-tubulin of chicken brain, an antibody to mouse IgG as, the second antibody and the radioactive iodinated protein A, and determination of the radioactivities of the bands on the nitrocellulose paper thus probed. The radioactivities were linearly proportional to the amounts of α- or β-tubulin from dark-grown Vigna mungo seedlings within a range of 4 to 56 ng or of 4 to 32 ng, respectively. This method was used to estimate the tubulin contents of several plant species using Vigna tubulin as a standard. α-Tubulin contents thus estimated were 25, 9, 19 and 11μg-equivalents of Vigna tubulin per mg protein for Vigna seedlings, Daucus suspension cells, Catharanthus suspension cells and Mougeotia cells, respectively. β-Tubulin contents of Vigna, Daucus, Catharanthus and Mougeotia cells were 29, 10, 13 and 5 μg-equivalents of Vigna tubulin per mg protein, respectively.
日本植物生理学会の論文