Duplexed, bead‐based competitive assay for inhibitors of protein kinases
Open Access
- 31 May 2007
- journal article
- research article
- Published by Wiley in Cytometry Part A
- Vol. 71A (7), 451-459
- https://doi.org/10.1002/cyto.a.20398
Abstract
Background: Many cellular signal transduction cascades have protein kinases as critical components. Small molecule protein kinase inhibitors can be effective as laboratory probes and drugs. Methods that allow two or more kinases to be evaluated simultaneously for inhibition by a small molecule would allow unequivocal tests of specificity and selectivity of action of the small molecule. Methods: Two hexahistidine‐tagged activin receptor‐like kinases were expressed in E. coli, purified, and bound to nickel beads. A fluorescent kinase ligand (F‐KL) that binds to the ATP‐binding site of these kinases with nanomolar affinity was developed. Binding of F‐KL with kinase on the bead made the beads bright, and inhibitors decreased the brightness. Results: A test panel of 17 nonfluorescent kinase inhibitors, spanning two orders of magnitude affinity for the kinases, gave Kd values for the kinases that correlated well with a fluorescence polarization assay. Results were obtained for the kinases in duplex, using an autosampler to send beads from a 96‐well plate to a flow cytometer in a format suitable for high throughput screening. Conclusions: Inhibitors of kinases can be measured in duplex in a high throughput format by flow cytometry, if a suitable fluorescent ligand is available. © 2007 International Society for Analytical CytologyKeywords
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