Determining protein-drug binding can be achieved through indirect and direct methods, each providing valuable insights into the interaction between proteins and drugs.

Indirect methods involve isolating the bound drug from its free form in biological samples such as blood, serum, or plasma. These techniques aim to measure the percentage of drugs bound to proteins. Equilibrium dialysis is a commonly used method where the free drug concentration at equilibrium is measured by separating the bound and unbound drug using a semi-permeable membrane. Dynamic dialysis, on the other hand, assesses the movement of the drug across a membrane to understand its binding characteristics.

Various techniques are employed to separate the protein-bound drug from its free form. Ultrafiltration involves using a defined molecular weight cutoff filter to separate the bound and unbound drug. Ultracentrifugation utilizes high-speed centrifugation to separate the protein-bound drug from the free drug in the sample. Gel filtration chromatography is another technique that separates molecules based on their size and molecular weight.

In contrast, direct methods estimate the characteristics of binding sites in pure protein solutions without separating the bound drug form. UV and fluorescence spectroscopy are commonly used in these methods to determine the concentration of protein-bound drugs. Researchers can gain insights into the binding characteristics by measuring the absorbance or fluorescence properties of the drug-protein complex. Ion-selective electrodes are also utilized to measure ion-protein binding and understand the interactions between drugs and specific ions.

Various plots are utilized to analyze binding data obtained from these methods. The direct plot provides information about the concentration of protein-bound drugs directly. The Scatchard plot, Klotz plot, or Lineweaver–Burk plot can provide insights into the affinity and capacity of the binding sites. The Hitchcock plot analyzes cooperative binding, where one drug molecule's binding affects subsequent molecules' binding.

Collectively, these methods provide valuable insights into the protein-drug interaction. By understanding the mechanisms and characteristics of protein-drug binding, researchers and healthcare professionals can optimize drug therapy, predict potential drug interactions, and ensure effective and safe pharmacological outcomes.