| Assay Method Information | |
| | Assay for Inhibition of KIF11 |
| Description: | The ATPase rate for Kif11 was monitored using an enzyme-coupled assay. Porcine brain microtubules (Cat #MT002) were purchased from Cytoskeleton (Denver, CO) and polymerized as per manufacturer's instructions and stored at 1 mg/ml at −80° C. GST-tagged Kif11/Eg5 (Cat #EG01-XL) and the Kinesin ELIPA Biochem Kit (Cat #BK060) was also purchased from Cytoskeleton. Taxol, microtubules, 7-methylthioguanosine (MESG), and purine nucleoside phosphorylase (PNP) were added to final concentrations of 15 mM, 0.05 mg/mL, 200 mM, 1U/m, 1 mM in EPLIA Reaction Buffer and allowed to incubate at room temperature for 15 minutes under rocking. Kif11 was then added at a final concentration of 0.025 mM and allowed to incubate at room temperature for an additional 15 minutes. Master mix (18.5 mL) was added to a black 384-well plate (Corning 3575), to which 0.5 mL of DMSO or compound were added to appropriate wells. Plates were incubated at room temperature for 2 hours. ATPase reactions were initiated by addition of 1 mM ATP and monitored by OD340 every 1 minute over the course of 2 hours. The rate of the reaction was taken from the linear part of the resulting curve. IC50 was determined by plotting inhibitor concentration v. ATPase rate. |
| Affinity data for this assay | |
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