J. Biol. Chem., Vol. 255, Issue 22, 10744-10751, 11, 1980

The quantitation of tubulin in neuroblastoma cells by radioimmunoassay

L Van de Water 3d and JB Olmsted

The amount of tubulin in a clone of neuroblastoma cells (Nb2a-AB-1) which undergoes microtubule-dependent neurite elongation has been determined. This clone undergoes shape differentiation without cessation of cell division, and both differentiated and nondifferentiated cells have the same cell cycle parameters. To quantitate tubulin pools, a radioimmunoassay was developed with a rabbit serum raised to sodium dodecyl sulfate-treated Tetrahymena axonemal tubulin. Using purified hog brain tubulin as labeled tracer and competitor, competition curves covering the range of 0.1 to 100 microgram/ml were obtained. Curves obtained using purified mouse brain or neuroblastoma tubulin as competitors were similar to those obtained with hog brain tubulin. The levels of competition obtained with hog tubulin, partially purified tubulin, or fixed microtubules were identical, indicating that the polymerization state of tubulin had no effect on measurements. Postmitochondrial supernatants derived either from cells grown in suspension culture (rounded morphology) or monolayer culture (neurite-bearing morphology) contained equivalent amounts of tubulin (3.6 +/- 0.5 pg/cell and 3.1 +/- 0.2 pg/cell, respectively); tubulin constituted 2.7% of the postmitochondrial supernatant protein in either cell type. These data indicated that cells utilizing microtubules primarily for mitosis or for cytoskeletal functions and mitosis show no net change in the total soluble tubulin pool.
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