This chapter illustrates the application of binding techniques to the study of drug-receptor interaction. It discusses the theory of binding and the methods used to quantify drug effect before the experimental prerequisites for good binding experiments. The methods used to detect and rectify nonequilibrium experimental conditions utilize the very methods used to quantify drug effect. Therefore, they must be understood before their application to optimize experimental conditions can be discussed. This chapter first presents what the experiments strive to achieve, and then explores the possible pitfalls of experimental design that may cause the execution to fall short of the intent. A direct measure of the binding of a molecule to a protein target can be made if there is some means to distinguish bound molecule from unbound and a means to quantify the amount bound. Historically, the first widely used technique to do this was radioligand binding. Radioactive molecules can be detected by observation of radioactive decay and the amount quantified through calibration curves relating the amount of molecule to the amount of radioactivity detected. An essential part of this process is the ability to separate the bound from the unbound molecule. This is done by taking advantage of the size of the protein versus the soluble small molecule. The protein can be separated by centrifugation, equilibrium dialysis, or filtration. Alternatively, the physical proximity of the molecule to the protein can be used.
