Method for identifying ligands that bind to cloned Gs- or Gi-coupled receptors
| Autor: | M. König, J.W. Marsg, J.S. Fink, M.J. Brownstein, L.C. Mahan |
|---|---|
| Rok vydání: | 1991 |
| Předmět: |
Agonist
chemistry.chemical_classification medicine.drug_class Cell Biology Transfection Biology Molecular biology Adenylyl cyclase Cellular and Molecular Neuroscience chemistry.chemical_compound Tissue culture Enzyme chemistry Biochemistry Cell culture Complementary DNA medicine Receptor Molecular Biology |
| Zdroj: | Molecular and Cellular Neuroscience. 2:331-337 |
| ISSN: | 1044-7431 |
| DOI: | 10.1016/1044-7431(91)90063-t |
| Popis: | We describe a fast, simple assay for testing ligands for binding to an unknown receptor after transfection of that receptor cDNA into tissue culture cells. The assay is based on a mouse L cell line (LVIP2.OZc) that contains a cyclic AMP responsive reporter construct and is performed in 96-well plates. If the appropriate agonist binds to a receptor clone coupled to G s proteins, activation of adenylyl cyclase produces cAMP which in turn induces the enzyme β-galactosidase in LVIP2.0Zc cells. β-Galactosidase activity is detected by staining cells with a chromogenic substrate. After cell lysis, incubation with o -nitrophenyl β- d -galactopyranoside (ONPG) results in a yellow color. Color development can be observed with the naked eye or read with a plate reader at 405 nm. Agonists that bind to G i -coupled receptors can be identified by inhibition of forskolin-induced expression of β-galactosidase. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|