3.3. In Vitro Tubulin Polymerization Inhibition Assay

The fluorescence-based in vitro tubulin polymerization assay was performed using the Tubulin Polymerization Assay Kit (BK011P, Cytoskeleton, USA) according to the manual. The tubulin reaction mix contained 2 mg/mL porcine brain tubulin (>99% pure), 2 mM MgCl₂, 0.5 mM ethylene glycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid (EGTA), 1 mM guanosine triphosphate (GTP), and 15% glycerol. First, a 96-well plate was incubated with 5 μL of inhibitors in different concentrations at 37 °C for 1 min. Then 50 μL of the tubulin reaction mix was added. Immediately, the increase in fluorescence was monitored by excitation at 355 nm and emission at 460 nm in a multimode reader.