Duplexed, bead-based competitive assay for inhibitors of protein kinases.
Cytometry A
; 71(7): 451-9, 2007 Jul.
Article
em En
| MEDLINE
| ID: mdl-17542028
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 K(d) 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.
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Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Ligação Competitiva
/
Inibidores de Proteínas Quinases
/
Citometria de Fluxo
/
Microesferas
Tipo de estudo:
Evaluation_studies
Limite:
Humans
Idioma:
En
Revista:
Cytometry A
Ano de publicação:
2007
Tipo de documento:
Article
País de afiliação:
Estados Unidos