Assay Method Information

Assay Name:  Radioligand Binding Assay
Description:  Saturation experiments. A competitive NaV1.7 inhibitor having a methyl group is tritiated. Three tritiums are incorporated in place of methyl hydrogens to generate [3H]compound. Binding of this radioligand is performed in 5 mL borosilicate glass test tubes at room temperature. Binding is initiated by adding membranes to increasing concentrations of [3H]compound in 100 mM NaCl, 20 mM Tris HCl, pH 7.4 buffer containing 0.01% w/v bovine serum albumin (BSA) for 18 h. Non-specific binding is determined in the presence of 1 μM unlabeled compound. After 18 h, the reactants are filtered through GF/C glass fiber filters presoaked in 0.5% w/v polyethylene imine. Filters are washed with 15 mL ice cold 100 mM NaCl, 20 mM Tris HCl, pH7.4 buffer containing 0.25% BSA to separate bound from free ligand. [3H]compound bound to filters is quantified by liquid scintillation counting.
Affinity data for this assay
 

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