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Assay Method Information |
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| | Binding Assay |
| Description: | The binding assay was performed by a filtration method in a 384 well format. Membranes at a final protein concentration of 6 μg/well were incubated with 125I-glucagon at 0.3 nM and in the presence of compound for 2 hours at room temperature in a total reaction volume of 40 μL per well. Assay buffer consisted of 50 mM HEPES, pH 7.4, 5 mM MgCl2, 1 mM CaCl2 and 0.2% BSA. 30 μL of the reaction was then transferred to PEI treated filter plates and followed by filter aspiration. Plates were then washed 5x and allowed to dry at room temperature overnight. The next day the bottom of the plate was covered with seal tape and scintillant was added. Total counts retained by the filters were quantified with a Top Count instrument. |
| Affinity data for this assay | |
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