3.8. Tubulin Polymerization Assay

Tubulin polymerization assays were conducted using the tubulin polymerization assay kit following the manufacturer’s instructions (Cytoskeleton, Inc., Denver, CA, USA). Briefly, 50 μL of 1.3 mg/mL tubulin (>99% pure) proteins in G-PEM buffer (consisting of 80 mmol/L PIPES 2 mmol/L MgCl₂, 0.5 mmol/L EGTA, 1 mmol/L GTP, and 15% glycerol at pH 6.9,) was placed in a quartz cuvette in the presence of the test agent (concentration: 0.25 µM). Polymerization was measured at every 3 s for 1 h using an Applied Photophysics Chirascan spectroflourimeter (excitation at 360 nm and emission at 420 nm) at 37 °C.