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© 1996 Springer-Verlag. Reproduced with permission.
(from Durso et al. 1996, Protoplasma 190: 141-150)
 Single immunolocalizations using conventional epifluorescence microscopy on footprints of carrot protoplasts lysed prior to fixation are shown. Anti-pp50 serum (1:300) revealed microtubule-like fluorescence (a), and footprint microtubules were detectable with serum even at 1:2000 (not shown). Preimmune serum (1:100) revealed little or no fluorescence (b), even at a higher concentration and longer photographic exposure times than a. Affinity-purified anti-pp50 antibodies (1:10) revealed microtubule-like fluorescence (c) more clearly than anti-pp50 serum (a), and footprint microtubules were detectable with affinity-purified anti-pp50 even at 1:40 (not shown). Affinity purification control eluate (1:1) off a strip of tubulin (as in Fig.1c) revealed little or no fluorescence (d), even at a higher concentration and longer photographic exposure times than c. The control eluate off random non-pp50 polypeptides (as in Fig.1c) produced similar results (not shown). These results indicate that pp50's microtubule-like immunolocalization depends on pp50-specific antibodies, and not on anti-tubulin contaminants or other components originally present in anti-pp50 serum. Bars = 4 um. |
