| Assay Method Information | |
| | ADP Hunter Plus Assay |
| Description: | Compounds were first tested in triplicates at 100 uM; hit compounds were further tested with a 10-point twofold serial dilution to confirm their activity and determine their IC50 values. S-Trityl-L-cysteine (STLC), a selective Eg5 inhibitor(J. Biol. Chem. 2006, 281:17559-17569; Mol. Cancer Ther. 2004, 3:1079-1090), was used as the control compound. Specifically, 20 uL of 15 mM PIPES (pH 7.0) containing 1 mM MgCl2, 50 nM MT, 20 uM paclitaxel, 200 uM ATP, 5% DMSO, 60 nM Eg5 proteins, and 1:2 serial dilutions of each individual compound starting from 1000 uM were added to each well of a 96-well plate. The plate was incubated at room temperature for 0.5 h, and then, the ADP Hunter Plus reagents were added. The plate was further incubated for 0.5 h and then read for fluorescence (ex.530/em.590) on Synergy 4 (BioTek, Winooski, VT, USA). |
| Affinity data for this assay | |
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