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Quantitation of Cellular Tubulin in Microtubules and Tubulin Pools by a Competitive ELISA

Quantitation of Cellular Tubulin in Microtubules and Tubulin Pools by a Competitive ELISA

D Thrower, M A Jordan, L Wilson

J Immunol Methods. 1991 Jan 24;136(1):45-51.

PMID: 1995712

Abstract:

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.

Chemicals Related in the Paper:

Catalog Number	Product Name	Structure	CAS Number	Price
IAR42411214	Alkaline Phosphatase Stabilizing Buffer			Price

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