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Expert researchers describe the key methods for detecting and analyzing proteins and protein motifs involved in targeting interactions, mostly from the perspective of intracellular signaling. The techniques include those designed to reveal the existence of targeting interactions and characterize their physico-chemical properties, to identify and isolate interacting proteins on the basis of the 'bait-prey' principle, to target domains, and to modify lipids. The methods are richly detailed for use by those researchers interested in defining and characterizing the protein-protein interactions that underlie targeting mechanisms in all areas of molecular cell biology.
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1 Surface Plasmon Resonance Measuring Protein Interactions in Real Time George Panayotou 1. Introduction Interactions between macromolecules play a central role in most biological processes. Their analysis in vitro can shed light on their role in the intact cell by providing valuable information on specificity, affinity, and structur&Lnctlon relationships. Significant progress in this respect has come with the advent, in the last few years, of commercially available biosensor technology (I). This has allowed the study of macromolecular interactions m real time, providing a wealth of high-quality binding data that can be used for kinetic analysis, affnity measurements, competition studies, and so on. A major advantage of biosensor analysis is that there is no requirement for labeling one of the interactmg components and then separating bound from free molecules--a fact that simplifies experimental procedures and provides more accurate measurements. The most successful and widely used procedure for blosensor analysis is based on the phenomenon of surface plasmon resonance (SPR), which occurs upon the interaction of monochromatic light with a gold surface, under conditions of total internal reflection (