Updates on contemporary protein binding techniques.

SUMMARY: Automated high-throughput techniques have become key to improving existing as well as new techniques associated with protein binding analysis. A wide variety of methods and experimental conditions are used for estimating protein binding as well as binding affinity, such as ultrafiltration and affinity chromatography. These methods rely either on the separation of a bound and free drug for subsequent conventional analysis or change in intrinsic parameters such as conformational properties of the protein. More recently developed techniques include surface plasmon resonance and solid-phase microextraction. Photoaffinity labeling, site-directed mutagenesis and x-ray crystallography are valuable techniques to identify the locations of binding sites on a protein. A new high-throughput assay based on the distribution of a drug among plasma water, plasma proteins, and solid-supported lipid membranes (Transil) has been reported to produce valid results, even for drugs that are strongly bound to plasma proteins. This new method may be suited for examining highly lipophilic drugs that adsorb onto surfaces due to their low solubility in aqueous media. Such a method may promote drug discovery and development for high-throughput determination of protein binding.