title: A high-throughput fluorescent polarization assay for nuclear receptor binding utilizing crude receptor extract.
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.
authors.affiliation: Leads Discovery, Dupont Pharmaceuticals Company, E357/1132, Experimental Station, Wilmington, Delaware 19880-0400,USA. siqi.lin@dupontpharma.com
authors.forename: Siqi
authors.initials: S
authors.lastname: Lin
authors.forename: Cindy L
authors.initials: CL
authors.lastname: Bock
authors.forename: Debra B
authors.initials: DB
authors.lastname: Gardner
authors.forename: Jeffery C
authors.initials: JC
authors.lastname: Webster
authors.forename: Margaret F
authors.initials: MF
authors.lastname: Favata
authors.forename: James M
authors.initials: JM
authors.lastname: Trzaskos
authors.forename: Kevin R
authors.initials: KR
authors.lastname: Oldenburg
mesh_terms: D000818:Animals; D001665:Binding Sites; D001667:Binding, Competitive; D002457:Cell Extracts / Q000737:chemistry; D003345:Corticosterone / Q000378:metabolism; D003907:Dexamethasone / Q000378:metabolism; D005454:Fluorescence Polarization / Q000379:methods*; D005938:Glucocorticoids / Q000378:metabolism; D006801:Humans; D006854:Hydrocortisone / Q000378:metabolism; D020128:Inhibitory Concentration 50; D007313:Insecta; D007700:Kinetics; D008024:Ligands; D011869:Radioligand Assay / Q000379:methods; D011965:Receptors, Glucocorticoid / Q000032:analysis* / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D015203:Reproducibility of Results; D018411:Spodoptera / Q000166:cytology
chemical_list: D002457:Cell Extracts; D005938:Glucocorticoids; D008024:Ligands; D011965:Receptors, Glucocorticoid; D011994:Recombinant Proteins; D003907:Dexamethasone; D003345:Corticosterone; D006854:Hydrocortisone