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.
Thrower
D
D
Department of Biological Sciences, University of California, Santa Barbara 93106.
Jordan
M A
MA
Wilson
L
L
D000918:Antibody Specificity; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D008870:Microtubules / Q000737:chemistry*; D014404:Tubulin / Q000032:analysis*
D014404:Tubulin