Figure 4.

Competition binding assay with colchicine and colchicine-504 ▲ and colchicine-646 ■ to tubulin (fluorescence polarization).a

^a Assay was carried out in black 384 well plate; colchicine was serially diluted from 100 µM to 0.04 µM in the presence of 20 µl buffer (10 mM, GTP¹⁹, 80 mM PIPES, 1 mM EGTA, 1 mM MgCl₂, pH 6.8), tubulin (10 µM), and colchicine-504 or colchicine-646 (10 nM or 100 nM, respectively). The binding was measured after 2h at 37°C¹⁹ using fluorescence polarization. The K_d values were obtained by fitting data to the following equation (y = min + (max − min)/1 + (x/Kd) Hill slope). Values are means of three experiments carried out in triplicate.