Quantitation and characterization of antibody binding to tubulin

A goat antiserum, raised to native hog brain tubulin, was characterized by conventional immunological techniques and by employing a method for precipitation of tubulin.anti-tubulin complexes with heat-inactivated Staphylococcus aureus. Antiserum dilution experiments indicated that antibodies to native tubulin were raised, and maximal binding was observed to microtubules fixed with 1 mM glutaraldehyde. Competition experiments, using iodinated fixed microtubules as tracer, demonstrated that equivalent binding occurred with microtubules at protein concentrations 100- to 1000-fold lower than those for monomeric tubulin. A rabbit antiserum raised to sodium dodecyl sulfate-treated axonemal tubulin was also characterized. The serum bound maximally (90%) to either sodium dodecyl sulfate-treated or native iodinated hog brain tubulin, and competition for antibody binding has been observed with tubulin from diverse sources (Tetrahymena pyriformis ciliary axonemes, Lytechinus pictus flagellar axonemes, and mouse neuroblastoma extracts). Using these two antisera, radioimmunoassays are being developed for quantitation of polymeric and total tubulin in cellular systems.