This protocol is intended for use in 96 well plates (1,200 μl wells) but it can similarly be applied to standard test tubes (Levant, 2007). D2, D3, and D4 dopamine receptors are members of the D2-like class of dopamine receptors. They can be studied using the radioligand [3H]-spiperone, which is an antagonist binding to D2, D3, and D4 receptors with comparable affinity. A saturation assay can be used to determine the affinity of a radioligand to a receptor (Kd) and to determine the total number of receptors present in the assay (Bmax). If saturation binding experiments are performed in the absence and presence of a fixed concentration of another, not radiolabeled ligand, it can also be determined whether the other ligand acts in a competitive manner. If the specific radioactivity is low (tritiated) relative to the affinity of the radioligand (< 1 nM), a high assay volume (≥ 1 ml) is required to avoid ligand depletion; this is of particular importance if a receptor source with high expression density is used (e.g. expressed recombinant receptors). To obtain reliable estimates of these parameters at least 6 different concentrations of radioligand must be tested, but particularly when a receptor is first detected in a given tissue or cell type a greater number of concentrations are helpful. The incubation time and temperature are chosen to allow formation of equilibrium between association and dissociation with the receptor for both radioligand and competitor. Each experiment can be divided into different steps such as assay preparation, membrane preparation, incubation, filtration, counting of the samples and data analysis. To minimize experimental error all assays are performed at least in duplicate. Radioligand dilutions should be prepared to cover the desired concentration range. Optimally these concentrations should cover both the high range (corresponding to 5-10x Kd and hence saturation of the receptor) and the low range (around Kd), so that both Kd and Bmax can reliably be estimated without undue extrapolation. At each radioligand concentration total and non-specific binding should be determined; the agent used for the definition of non-specific binding (NSB) should be chemically (different family) and physically (avoid combination of two lipophilic compounds) distinct from the radioligand to avoid artifacts. For discussion of specific benefits of chosen assay conditions see van Wieringen et al. (2013) (copy can be obtained from the author).
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