Detection of tubulin-binding proteins by an overlay assay

A blot overlay technique to detect tubulin-binding proteins has been developed. It involves the fractionation of the putative tubulin-binding proteins by gel electrophoresis, their transfer to nitrocellulose paper, and the incubation of the nitrocellulose sheet with purified tubulin. The proteins which bind tubulin are recognized by monoclonal anti-tubulin antibodies. Proteins which associate with microtubules through a direct interaction with tubulin are easily detected by this procedure, which has been tested for both known microtubule-associated proteins and newly reported tubulin-binding proteins.