3.3. Tubulin Polymerization Assay

Microtubule assembly was studied using a tubulin polymerization assay kit (Cat. #BK006P, Cytoskeleton Inc., Denver, CO, USA) which was used according to the manufacturer’s instructions [42,43]. The polymerization was monitored using a FLUOstar Omega multi-mode microplate reader (BMG LABTECH, Offenburg, Germany). The final buffer concentrations for tubulin polymerization contained 80 mM PIPES (piperazine-N,N′-bis(2-ethanesulfonic acid)sequisodium salt), pH = 6.9, 2 mM MgCl₂, 0.5 mM EGTA (ethylene glycol-bis (β-amino-ethyl ether) N,N,N′,N′-tetraacetic acid, 1 mM GTP and 10.2% glycerol. Test compounds were added in one single concentration (10 μM) and then all compounds except the purified tubulin were warmed to 37 °C. The reaction was initiated by the addition of tubulin to a final concentration of 3.0 mg/mL. Paclitaxel and phenstatin were used as positive controls under the same conditions. The absorbance was measured at 340 nm for 1 h at 1 min intervals at 37 °C.