Radioligand Binding Assays

Ligand binding assays are an analytical procedure, whose method relies on the binding of specific ligand molecules to receptors, antibodies or other macromolecules (Hulme et al., 2010). The analysis of radioligand binding assays are based on a simple model, called the law of mass action, which assumes that binding to the receptors are reversible. The binding to the receptors occurs when the ligand and the receptor collide by diffusion and when the collision has enough energy and the correct orientation. Once binding has occurred, the receptor and ligand remain bound together for some time but there is a high probability of dissociation as the receptor doesn't "know" how long it has been bound to the ligand. After dissociation, the receptor and ligand are the same as they were before binding. Equilibrium is only reached when the rate at
Carbachol is a cholinergic agonist as it binds to muscarinic and nicotinic receptors for acetylcholine and stimulates them, causing a smooth muscle contraction. Atropine is non-selective muscarinic receptor antagonist. It is a reversible competitive antagonist of the muscarinic acetylcholine receptors and blocks the effects of carbachol. (Rang et al., 2012). 3H-NMS is a high affinity antagonist of the muscarinic acetylcholine receptor. The Schild analysis is used for studying the effects of antagonists and agonists on the cellular response caused by a receptor or on ligand-receptor binding. The Schild analysis can be used in ligand binding assays as it can reveal if there are more than one type of receptor, as well as, if the experiment was done wrong as the system will not reached equilibrium. The aim of the experiment was to determine the equilibrium dissociation constants of the ligands, carbachol and atropine at the human muscarinic M3 receptor from a stable recombinant Chinese hamster ovary, using the radioligand saturation and competition binding