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 competition experiment is usually performed to determine the affinity of a compound for a receptor. If multiple subtypes or states of the receptor are present and the competing compound differentiates them, a competition binding experiment can quantify the relative contribution of the two subtypes or states; while resolution of more than two subtypes or states is theoretically possible, in practical terms it is almost never feasible. Thus, radioligand binding to a receptor is quantified in the presence of various concentrations of the unlabelled compound of interest. The concentration of the radioligand in a competition study should be about 2-3 its Kd value as determined in saturation binding; this will allow a sufficient occupancy of the receptor to obtain a strong signal and at the same time avoid that competition becomes too difficult due to high radioligand concentration. The incubation time and temperature are chosen to allow formation of equilibrium between association and dissociation with the receptor for both radioligand and competitor. Of note, a simple competition experiments does not necessarily prove a competitive nature of the interaction between unlabelled drug and receptor. 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). The number of required competitor concentrations depends on the goal of the experiment. If only a rough estimate of antagonist potency is required, 1-2 concentration per log increment will be sufficient. However, if it is the aim to test for possible subtypes or states of the receptor, 3-5 concentrations per log increment are needed. If possible, the lowest competitor concentrations in the assays should not cause any detectable inhibition, whereas the highest concentrations should completely abolish specific binding. 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. Additionally, duplicates of total binding and non-specific binding should be included in the assay; 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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