To characterize the binding of coumarin-30 to tubulin in the presence or absence of additional ligands, 1.5 µM coumarin-30 was mixed 1:1 with solutions containing tubulin concentrations ranging from 4 nM to 100 µM in BRB-80-T buffer (80 mM K-PIPES, 1 mM MgCl2, 1 mM EGTA, 0.005% Tween-20, pH 6.8). Extra ligands were added in excess, typically at the concentration of 100 μM.