4.13. [3H]-Colchicine Tubulin-Binding Assay

The tubulin was prepared from bovine brain as previously described [40]. Pure tubulin (3 µM final concentration) in cold BRB80 buffer was mixed at 4 °C with a mix of [³H]-colchicine (82.6 Ci/mmol, Perkin-Elmer, #NET189250UC, 50 nM final concentration, Courtaboeuf, France) and the competitor Carba1 (100 µM final concentration) in a final volume of 200 µL. Following a 30-min incubation at 30 °C, the samples were deposited onto 50 µL of presedimented DEAE Sephadex A25 in BRB80 buffer. All subsequent steps were carried out at 4 °C. Samples were incubated for 10 min with continuous shaking to ensure quantitative binding of tubulin to the gel. Following centrifugation (2400 g, 4 min), supernatants were discarded and the pellets containing the bound molecule-tubulin complexes were washed four times with 1 mL of BRB80 buffer. Pellets were incubated for 10 min with 500 µL of ethanol to solubilize the tubulin-bound tritiated colchicine and 400 µL aliquots of the ethanol solutions were transferred to 5 mL of Ultima Gold scintillant (Perkin-Elmer) for determination of radioactivity.