Comparison [3H]-flumazenil binding parameters in rat cortical membrane using different separation methods, filtration and centrifugation

Radioligand receptor binding assays are a common method to evaluate the affinity of newly synthesized benzodiazepine ligands for the receptor. [3H]-flumazenil is an antagonist of benzodiazepine receptors and is generally used as a radioligand. In this study, the binding parameters of [3H]-flumazenil to rat cortical membranes were evaluated using two separation methods: filtration with GF/C filters and centrifugation. Additionally, the effects of vacuum pressure, exposure time to the cocktail, and geometry on the filtration method were studied. The binding parameters of [3H]-flumazenil (Kd and Bmax) were determined through saturation studies using two methods. The results from this study showed that the filtration method is time consuming and requires more steps to be completed. Because filtration causes partial elution of bound [3H]-flumazenil into the liquid scintillation cocktail, the results are not reproducible, which result in inaccurate estimation of the binding parameters. The centrifugation method in contrast to filtration is straightforward and produces reproducible as well as reliable results, all of the steps are performed in a single polypropylene tube, which eliminates the loss of tissue and avoids other systematic errors associated with transfer and handling.