VIRTUALLY EVERY cellular process involves the binding of a protein to
another protein, a DNA sequence, a RNA sequence, a peptide, or an endogenous small
... [Show More] molecule. Studying the binding between these binding partners plays a important role in elucidating the cellular processes, such as transcription, translation, metabolism, or signal transduction pathways. In addition, the study of binding between synthetic
small molecules to target proteins is a critical step in small-molecule drug discovery.
Many methods have been developed to study the protein binding processes. Some of the methods are only applicable to the interaction between a large protein and a smaller binding partner (e.g., protein polarization assays and equilibrium dialysis) while other
methods do not have this limitation (e.g., surface plasmon resonance and ELISA). The most desirable method should be able to study the binding partners with minimum perturbation of the system and generate robust signals over a large range of protein concentrations and with adequate temporal resolution. Because of the need to quantify the components in the binding system, many binding assays either perturbed the equilibrium of binding by separating the binding components or chemically modified some of the binding partners. Few methods, such as the well-established equilibrium
dialysis method, have negligible perturbation to the binding system. However, the
throughput in an equilibrium dialysis study is too slow to be useful for a large number
of test compounds. In this chapter, we will discuss the commonly used biochemical
technologies to study binding processes with focus placed on the underlying
principles, practices, and limitations. Cell-based assays to study protein–protein
interactions in vivo [such as the yeast two-hybrid systems and protein fragment
complementation assay (PCA) method] are beyond the scope of this chapter and will not be discussed. The basic principles and general equations describing binding
process are discussed in Chapter 3. [Show Less]