To verify this, we performed measurements of coumarin-30 and tubulin in the presence of each of the following established binders of the colchicine site: colchicine, nocodazole, podophyllotoxin, combretastatin A-4, 32 ELR510444 33 (Fig. ). Each ligand was added at a concentration exceeding its expected dissociation constant to ensure association with a significant fraction of tubulin. All ligands displayed the expected C-MST signals above the control level (Fig. ,f, Fig. ), quantitatively similar to that of free coumarin-30 (Fig. ). To test the specificity of the assay, we used several common laboratory reagents (IPTG, glutathione, pepstatin A, PMSF), which were not expected to associate with tubulin. The signals in the presence of these compounds were no different from the control level (Fig. , Fig. ).