Quantitation of cellular tubulin in microtubules and tubulin pools by a competitive ELISA.

A comprehensive method is described for isolating microtubules from cultured mammalian cells and quantitating the tubulin content of both the microtubules and total cellular tubulin pools with a competitive enzyme-linked immunosorbent assay (ELISA). The microtubule isolation procedure involves detergent lysis of cells in a microtubule stabilizing buffer, high speed centrifugation to collect the cytoskeletons, and subsequent solubilization of tubulin from microtubule-containing pellets. The competitive immunoassay involves preincubating an anti-tubulin monoclonal antibody with an unknown quantity of tubulin in cell extracts or solubilized microtubules to quantitatively reduce the antibody available to bind to a tubulin-coated microtiter plate. Binding of remaining antibody to the microtiter plate is measured spectrophotometrically using an alkaline phosphatase-conjugated secondary antibody. Quantitation is accomplished by comparison with a known quantity of bovine brain tubulin.