| Assay Method Information | |
| | Radioligand Binding Assay |
| Description: | The compounds of invention were solubilized and carried through serial dilutions using 100% DMSO. Aliquots from the compound serial dilutions were further diluted 25 fold into assay buffer (50 mM Tris-Cl pH 7.5, 5 mM MgCl2, 0.005% Triton X-100) and transferred (volume 50 ul) into 96 well assay plates. [125I]-CGRP (GE Healthcare or Perkin-Elmer) was diluted to 72 pM in assay buffer and a volume of 50 ul was added to each well. SK-N-MC membranes were thawed, diluted in assay buffer with fresh 0.1% mammalian protease inhibitor cocktail (Sigma), and re-homogenized. SK-N-MC homogenate (7 ug/well) was added in a volume of 100 ul. The assay plates were then incubated at room temperature for 2 h. Assays were stopped by addition of excess cold wash buffer (50 mM Tris-Cl pH 7.5, 0.1% BSA) immediately followed by filtration over glass fiber filters (Whatman GF/B) previously soaked in 0.5% PEI. Non-specific binding was defined with 1 uM beta-CGRP (Bachem). Protein bound radioactivity was determined using a gamma or scintillation counter. The resulting data was analyzed using a four parameter competitive binding equation (XLfit v2.0) and the IC50 was defined as the concentration of a compound of invention required to displace 50% of radioligand binding. Final assay concentration of [125I]-CGRP was 18 pM. The mean Kd for [125I]-CGRP is 25.4 pM. All compounds of invention were evaluated in at least two separate experiments. |
| Affinity data for this assay | |
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