Antisera against electrophoretically purified tubulin stimulate colchicine-binding activity.

Abstract

Several rabbit antisera have been prepared against reduced and alkylated, electrophoretically purified tubulin isolated from chick brain. These antisera give a single precipitin line in Ouchterlony double diffusion plates when tested against partially purified tubulin, and label specifically microtubule-and tubulin-containing structures, such as mitotic spindles, cilia, and vinblastine-induced crystals, in a variety of cells. The same antisera also display the unique ability to stimulate the colchicine-binding activity of tubulin preparations from chick brain and Chinese hamster ovary tissue culture cells. This specific stimulation of colchicinebinding activity is also obtained with the gamma globulin fractions purified by ammonium sulfate precipitation of these antisera.Intensive effort has been directed at characterizing the distribution, ultrastructure, and mode of action of microtubules; their assembly from tubulin subunits; and their response to various agents, for example, temperature, pressure, and the plant alkaloids colchicine and vinblastine (1-5). Recent efforts have centered upon the raising of specific antisera (8-12) so that microtubule structure can be studied immunologically. The antisera have been prepared in different ways against intact microtubules or tubulin, using for immunization preparations having various degrees of purity obtained from a number of sources, including vinblastine-induced paracrystals, flagellar outer doublets, and tubulin isolated from brain. By the fluorescein-labeled antibody technique, the antisera have been observed to stain specifically microtubule-and tubulin-containing structures such as vinblastine paracrystals (6), mitotic spindles (6, 7, 10), and fibrous networks in the cytoplasm (11). Whether or not these antisera affect the rather specific binding of colchicine to tubulin (3, 4) has not been reported.We report here the preparation and initial characterization of antisera produced against electrophoretically purified, reduced, and alkylated tubulin from chick brain. bulin bands were located by scanning the gels at 280 nm, they were cut out and the tubulin was eluted and used for immunization. About 30% of the original amount placed on the preparative gels was recovered. When the recovered tubulin was tested by analytical gel electrophoresis, only the two tubulin bands were detected.Antisera. Each of several rabbits was injected subcutaneously with a total of 1 mg of the reduced-alkylated, electrophoretically purified tubulin in complete Freund's adjuvant, over a period of 6 weeks. A detailed method for preparing the antisera will be published elsewhere (manuscript submitted for publication). The antisera obtained were tested by Ouchterlony double diffusion in 1% agarose gels at 40 for 24 hr. Control sera were obtained from the same rabbits prior to immunization.Cells. Mouse embryo fibroblasts, HeLa cells, and CHO cells were grown on coverslips in a-minimal Eagle's medium and 10% fetal calf serum (Flow Laboratories). To induce crystal formation, 10 ,ug/m...

An Immunocytochemical Study of the Human Odontoblast Process using Antibodies against Tubulin, Actin, and Vimentin

“…The microtubules, intermediate filaments, and microfilaments are the major components of the cytoskeleton in eukaryotic cells, and have not been shown to exist extracellularly in viable cells (Aubin et al, 1976;for reviews, see Aubin, 1981, also Weber and Osborn, 1981).…”

Immunoflourescent staining of cytoplasmic and spindle microtubules in mouse fibroblasts with antibody to tau protein.

“…The antiserum obtained has been previously characterized (20,21) and reveals specific staining of mitotic spindles and other microtubule-containing structures.…”

“…Preimmune sera were obtained from the same animals prior to immunization. The preparation of antiserum to tubulin has been previously described (20).…”

New Immunological Approaches to Studying the Odontoblast

“…This is in keeping with the generalized structural characteristics of numerous other processes, including those of osteocytes (Holtrop and Weinger, 1972) and the axons of neuronal cells (for review, see Lasek and Brady, 1981). Finally, after approximately 20 years of detailed ultrastructural analysis (Porter, 1966;Ishikawa et al 1969;for review, see Porter et al, 1983), and approximately ten years of investigation by immunofluorescence (Lazarides and Weber, 1974;Aubin et al, 1976;for review, see Aubin, 1981;Weber and Osborn, 1981), cytoskeletal elements have not been shown to penetrate extra~el lularly in viable cells. Thus, we have commenced by steppmg back from the functional and structural complexities and uniqueness of the odontoblast and its process, to apply techniques designed to delineate cyto-architectural components thought to be ubiquitous in all cells of higher organisms.…”

“…As background. however, to the first specific application discussed here, it ĩ best~o dig~ess briefly to explain that antisera hav~been~repared m rabbits agam.st tubulin the major subunit protem of microtubules, The tubuhn had been electrophoretically-purified to reduce the number of contaminating 'polypeptides that typically co-purify with tubulin (Aubin et al, 1976;McKeithan and Rosenbaum, 1984). Antibodies specific for tubulin were affinity-purified from the antisera by chromatography on Sepharose 4B to which tubulin had been covalently coupled (Weber et al, 1975;Aubin, 1981).…”