Tubulin Polymerization Assay

Tubulin polymerization assay was performed using a kit, and with slight modification of the manufacturer’s (Cytokeleton Inc.) protocol. The recommendation for the in vitro tubulin polymerization assay kit is to use tubulin at 2 mg/ml in fluorescence buffer (80 mM PIPES pH 6.9, 2.0 mM MgCl₂, 0.5 mM EGTA, 10 μM fluorescent reporter) + 1 mM Guanosine-5′-triphosphate (GTP) in black 96-well plates. As R-roscovitine is a synthetic GTP analog, a comparable amount of R-roscovitine was used: 0.2 mM or 0.4 mM when used alone or 0.4 mM when used together with 0.5 mM GTP. For control, Dimethyl sulfoxide (DMSO) and ddH₂O were used instead of R-rsocovitine and GTP, respectively. The kinetics of tubulin polymerization was measured up to 60 min at 37°C using a spectrophotometer (SpectraMax M2, Molecular Devices; Softmax Pro software). Excitation and emission wavelengths were set at 355 nm and 460 nm, respectively.