Monoclonal antibodies specific to plant tubulin (original) (raw)
Related papers
BMC Plant Biology, 2010
Background: The function of the cortical microtubules, composed of αβ-tubulin heterodimers, is linked to their organizational state which is subject to spatial and temporal modulation by environmental cues. The role of tubulin posttranslational modifications in these processes is largely unknown. Although antibodies against small tubulin regions represent useful tool for studying molecular configuration of microtubules, data on the exposure of tubulin epitopes on plant microtubules are still limited. Results: Using homology modeling we have generated an Arabidopsis thaliana microtubule protofilament model that served for the prediction of surface exposure of five β-tubulin epitopes as well as tyrosine residues. Peptide scans newly disclosed the position of epitopes detected by antibodies 18D6 (β1-10), TUB2.1 (β426-435) and . Experimental verification of the results by immunofluorescence microscopy revealed that the exposure of epitopes depended on the mode of fixation. Moreover, homology modeling showed that only tyrosines in the C-terminal region of β-tubulins (behind β425) were exposed on the microtubule external side. Immunofluorescence microscopy revealed tyrosine phosphorylation of microtubules in plant cells, implying that β-tubulins could be one of the targets for tyrosine kinases.
Tubulin assembly probed with antibodies to synthetic peptides
Journal of Molecular Biology, 1990
Antibodies to synthetic peptides from the u and #?-tubulin sequences were employed to study zones of this protein active in microtubule assembly. In purified calf brain tubulin, six short sequences, selected according to their hydrophilicity and conservation, were found to be accessible to their affinity-purified immunoglobulin G (IgG) antibodies, in a competition radioimmunoassay performed under non-assembly native conditions. This indicated that the six sequences arc exposed on the surface of the tubulin UP heterodimer.
The Plant Journal, 1993
The knowledge of higher plant microtubule-associated proteins (MAPs) remains limited to a few examples that illustrate essentially their binding properties to preformed microtubules as described in carrots. Using taxof-stabilized microtubules a putative MAP-enriched fraction has been isolated in maize cultured cell extracts, one of these polypeptides is immunologically related to neural tau. At present, these proteins are being characterized by co-assembly assays that were not possible before. Similar experiments were done also in a heterologous system using brain tubulin. Three polypeptides out of seven that constituted the MAP fraction were found to co-assemble specifically with tubulin subunits of both origins. Their apparent molecular weights are 67, 83 and 125 kDa. A twodimensional gel immunoblot of the 83 kDa polypeptide with tau antibodies revealed one major spot. Polypeptides were quantiated by scanning the gels. These results shed light on the present debate on higher plant MAPs and their potential activity in the regulation of microtubule assembly and function in the higher plant cell.
Post-translational modifications and multiple tubulin isoforms in Nicotiana tabacum L. cells
Planta, 1997
Distribution of post-translationally modified tubulins in cells of Nicotiana tabacum L. was analysed using a panel of specific antibodies. Polyglutamylated, tyrosinated, nontyrosinated, acetylated and Δ2-tubulin variants were detected on -tubulin subunits; polyglutamylation was also found on -tubulin subunits. Modified tubulins were detected by immunofluorescence microscopy in interphase microtubules, preprophase bands, mitotic spindles as well as in phragmoplasts. They were, however, located differently in the various microtubule structures. The antibodies against tyrosinated, acetylated and polyglutamylated tubulins gave uniform staining along all microtubules, while antibodies against nontyrosinated and Δ2-tubulin provided dotlike staining of interphase microtubules. Additionally, immunoreactivity of antibodies against acetylated and Δ2-tubulins was strong in the pole regions of mitotic spindles. High-resolution isoelectric focusing revealed 22 tubulin charge variants in N. tabacum suspension cells. Immunoblotting with antibodies TU-01 and TU-06 against conserved antigenic determinants of -andtubulin molecules, respectively, revealed that 11 isoforms belonged to the -subunit and 11 isoforms to thesubunit. Whereas antibodies against polyglutamylated, tyrosinated and acetylated tubulins reacted with several -tubulin isoforms, antibodies against nontyrosinated and Δ2-tubulin reacted with only one. The combined data demonstrate that plant tubulin is extensively posttranslationally modified and that these modifications participate in the generation of plant tubulin polymorphism.
Monoclonal antibodies that recognize discrete forms of tubulin
Proceedings of the National Academy of Sciences, 1982
Anti-tubulin antibodies secreted by plasmacytoma NSI-spleen cell hybrids were detected by an indirect binding assay. Different antibodies bound to different combinations of the tubulins as resolved by isoelectric focusing. Two monoclonal antibodies (TUB 2.1 and TUB 2.5) labeled only (i) the tubulin band on a polyacrylamide electropherogram and (ii) f3-tubulins as resolved by isoelectric focusing. The fraction that was specifically bound and eluted from antibody affinity columns was enriched in 1-tubulins as compared with a-tubulins, suggesting the possibility of some soluble tubulin homodimers and a3,-heterodimers. Double labeling experiments were used to show that all detectable microtubules contained (3-tubulin.