Biotin labeled tubulin

This assay was performed for competition binding to the colchicine and vinblastine binding sites on tubulin and carried out in a 96-well plate. Biotin-labeled tubulin (Cytoskeleton, Denver, CO) was dissolved in G-PEM buffer (80 mM PIPES pH 6.8, 2 mM MgCl₂, 0.5 mM EGTA, 1 mM GTP and 10% glycerol) to a final concentration of 0.8–1 mg/ml. [³H]colchicine or [³H]vinblastine (final concentration of 50 nM and 100 mM, respectively), 50 µM of SRF and 1.0 µg of Biotin-labeled tubulin was mixed in a final reaction volume of 100 µl of G-PEM buffer and incubated for 45 min at 37°C. Streptavidin SPA beads (80 µg/well) was added to each reaction mix and incubated for an additional 30 min at 4°C. The radioactive counts were measured using a Wallac Microbeta scintillation counter (PerkinElmer, Waltham, MA). For control reactions, SRF was omitted from the reaction mix.