A sensitive capillary electrophoretic method was developed to detect the presence of α-tubulin, a microtubular cytoskeletal component, in isolated nuclear preparations. These preparations are treated with anti-α-tubulin primary mouse antibodies and then stained with a fluorescently labeled anti-mouse IgG antibody. The stained preparation is then analyzed by capillary electrophoresis with laser-induced fluorescence detection, a technique that allows for sensitive detection of fluorescently labeled species. Using this method, it is feasible to count individual subcellular aggregates containing α-tubulin (SATs), estimate the number of α-tubulin molecules per SAT, determine the cumulative intensity of all SATs as an estimate of the relative level of α-tubulin in a preparation, and obtain their apparent electrophoretic mobility distribution. The method was validated by comparing SATs from untreated cells with those from colchicine-treated cells. Since colchicine is a microtubule-disrupting agent, treatment reduced the number of SATs per cell as well as the cumulative intensity of all SATs in a preparation. In contrast, the apparent electrophoretic mobility distribution was not influenced by colchicine treatment, suggesting that this parameter is not strongly dependent on the α-tubulin content. Given the zeptomolar sensitivity of laser-induced fluorescence detection and the widespread availability of antibodies, the approach used here represents an improvement in the detection of cytoskeletal impurities in subcellular fractions.
Bibliographical noteFunding Information:
This work was supported by NIH Grants GM61969 (EA) and GM49928 (KMF). E.A. also acknowledges support from NIH K02-AG21453.
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- Capillary electrophoresis