| Assay Method Information | |
| | Scintillation Proximity Assay |
| Description: | Receptor SPA binding assays were performed in white 96-well plates in a total volume of 200 mI per well. Freeze dried analogues were dissolved in 80% dimethyl sulfoxide, 20% H20 and serial dilutions (1:10) were performed in binding buffer (20 mM Tris-HCI pH 7.4, 5 mM MgAc2, 2 mM EGTA and 0.1% ovalbumin); the final assay concentrations ranging from 1 mM to 1 pM. Analogues and 7.5-50 pg of CRFi receptor membranes/well or 0.5 pg of CRF2 receptor membranes/well were added to assay plates. A mix of wheat germ agglutinin coated SPA beads (PerkinElmer) and [125l]-Sauvagine (PerkinElmer), both in binding buffer, was added to yield 0.4 mg beads/well and 50,000 cpm of radio ligand per well. Plates were sealed and incubated at 25 °C for 2 hours in a plate shaker set at 400 rpm and thereafter centrifuged at 1500 rp for 10 minutes. SPA plates were let to stand at room temperature for about 16 hours prior to reading on microplate scintillation counter. Displacement of radioligand was measured as reduction in luminescence, and IC50 values were calculated by nonlinear regression analysis of four parameter sigmoidal dose-response curves. |
| Affinity data for this assay | |
|---|---|
| If you find an error in this entry please send us an E-mail | |