A capillary electrophoretic method for monitoring the presence of alpha-tubulin in nuclear preparations.
A sensitive capillary electrophoretic method was developed to detect the presence of alpha-tubulin, a microtubular cytoskeletal component, in isolated nuclear preparations. These preparations are treated with anti-alpha-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 alpha-tubulin (SATs), estimate the number of alpha-tubulin molecules per SAT, determine the cumulative intensity of all SATs as an estimate of the relative level of alpha-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 alpha-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.
Gunasekera
Nilhan
N
Department of Chemistry, University of Minnesota, 207 Pleasant St SE, Minneapolis, MN 55455, USA.
Xiong
Guohua
G
Musier-Forsyth
Karin
K
Arriaga
Edgar
E
D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry; D002458:Cell Fractionation; D002467:Cell Nucleus / Q000737:chemistry*; D003078:Colchicine / Q000494:pharmacology; D019075:Electrophoresis, Capillary; D005453:Fluorescence; D005456:Fluorescent Dyes / Q000737:chemistry; D006074:Gout Suppressants / Q000494:pharmacology; D007834:Lasers; D051379:Mice; D008870:Microtubules / Q000737:chemistry / Q000187:drug effects; D012680:Sensitivity and Specificity; D013050:Spectrometry, Fluorescence / Q000379:methods; D014404:Tubulin / Q000737:chemistry*
D000911:Antibodies, Monoclonal; D005456:Fluorescent Dyes; D006074:Gout Suppressants; D014404:Tubulin; D003078:Colchicine