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Plant and Cell Physiology, 1986, Vol. 27, No. 2 273-283
© 1986

Article

Establishment of a Sensitized Immunoblotting Method for Measuring Plant Tubulin Content

Hiroo Fukuda and Nobuo Iwata

Department of Biology, Faculty of Science, Osaka University Toyonaka, Osaka 560, Japan

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 {alpha}- or ß-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 {alpha}- or ß-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. {alpha}-Tubulin contents thus estimated were 25, 9, 19 and 11 µg-equivalents of Vigna tubulin per mg protein for Vigna seedlings, Daucus suspension cells, Catharanthus suspension cells and Mougeolia cells, respectively. ß-Tubulin contents of Vigna, Daucus, Catharanthus and Mougeotia cells were 29, 10, 13 and 5 µg-equivalents of Vigna tubulin per mg protein, respectively.

(Received August 6, 1985; Accepted December 5, 1985)
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