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Tubulin polymerization assay

AM Alborz Mazloomian
SA Shinsuke Araki
MO Momoko Ohori
AE Amal M. El-Naggar
DY Damian Yap
AB Ali Bashashati
SN Shoichi Nakao
PS Poul H. Sorensen
AN Atsushi Nakanishi
SS Sohrab Shah
SA Samuel Aparicio
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The compounds (T-595, T-598 and cochicine control) were screened at varying concentrations with 2.0 mg ml−1 tubulin in Polymerization Buffer in 80 mM PIPES buffer pH 6.9, 2 mM MgCl2, 0.5 mM EGTA, 1 mM GTP, 10 μM DAPI, 15% v/v glycerol, and 2% v/v DMSO from the compound solvent by the manufacturer (Cytoskeleton Inc, CO, USA). Polymerization was followed by fluorescence enhancement due to the incorporation of a fluorescent reporter into microtubules as polymerization occurs. The Vmax (maximum slope) values for the tubulin polymerization curves were compared against the control to create a percent of control value, which were plotted against Log10 each concentration. Curves were fit by non-linear regression to a 4-parameter sigmoidal dose-response equation to determine the IC50 for each compound.

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