| Assay Method Information | |
| | Binding Assay |
| Description: | The binding assays are carried out using a Scintillation Proximity Assay (Amersham) with WGA beads previously blocked with 1% fatty acid free BSA (ICN). The binding buffer contains 25 mM Hepes, pH 7.4, 2.5 mM CaCl2, 1 mM MgCl2, 0.1% fatty acid free BSA, (ICN), 0.003% tween-20, and Roche Complete Inhibitors without EDTA. Glucagon is dissolved in 0.01 N HCl at 1 mg/mL and immediately frozen at −80 degrees C. in 30 μl aliquots. The glucagon aliquot is diluted and used in binding assays within an hour. Test compounds are dissolved in DMSO and serially diluted in DMSO. 10 ul diluted compounds or DMSO is transferred into Corning 3632, opaque clear bottom assay plates containing 90 μl assay binding buffer or cold glucagon (NSB at 1 μM final). 50 μl of 1-125 glucagon (0.15 nM final in reaction), 50 μl of membranes (300 μg/well), and 40 μl of WGA beads (150 mgs/well) are added, covered, and mixed end over end. |
| Affinity data for this assay | |
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