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Anal Biochem ; 300(1): 15-21, 2002 Jan 01.
Article in English | MEDLINE | ID: mdl-11743686

ABSTRACT

A homogenous high-throughput assay has been developed to measure the binding between nuclear receptors and test compounds. This assay applies a fluorescence polarization (FP) detection method using human glucocorticoid receptor (GR) as a model system. Crude receptor extract, which requires no additional purification, is used in the assay. The binding conditions (i.e., DMSO tolerance, temperature, stability, and variability) have been investigated and validated. At the optimized conditions, a signal-to-background ratio of 2:1 and a Z'-factor of 0.7 was achieved in a 384-well format. Several known strong and weak GR ligands have been evaluated in this system. Possible interference of fluorescent compounds and methods to identify false positives are also discussed. This FP-based assay system can potentially be used for many soluble nuclear receptors in high-throughput binding assays.


Subject(s)
Fluorescence Polarization/methods , Receptors, Glucocorticoid/analysis , Animals , Binding Sites , Binding, Competitive , Cell Extracts/chemistry , Corticosterone/metabolism , Dexamethasone/metabolism , Glucocorticoids/metabolism , Humans , Hydrocortisone/metabolism , Inhibitory Concentration 50 , Insecta , Kinetics , Ligands , Radioligand Assay/methods , Receptors, Glucocorticoid/metabolism , Recombinant Proteins/metabolism , Reproducibility of Results , Spodoptera/cytology
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