Fluorescence methods ligand-receptor binding

Affinity probe capillary electrophoresis (APCE) uses a fluorescently labeled ligand to bind a receptor. Separation of the bound and free affinity probe has been achieved with CE with LIE detection. One advantage of the rapid CE method is that if the separation is rapid compared to the dissociation time, affinity constants can be determined. Competition experiments can be performed to observe the conditions of complex formation. For example, a fluorescent GTP analog was used as an affinity probe and G protein receptor subunits as the ligand. The complex and free fluorescent probe were separated within 20 s and Ras-like G protein complexes could be separated in less than 15 s. The rapid separations allowed kinetics of binding to be studied. 

In some instances, flow cytometry assays are a superior alternative to conventional procedures for the determination of equilibrium binding constants (Stein et al., 2001). In contrast to assays that employ radiolabelled ligands, which measure population mean values for binding constants, flow cytometry methods can measure those values in individual cells, revealing heterogeneity in receptor expression within a population of cells or membrane vesicles. Furthermore, small samples can be characterized in a short period of time (hours). This approach to receptor-binding analysis may be limited only by the availability of a properly characterized fluorescent ligand. 

Real-time spectroscopic methods can be used to measure the binding, dissociation, and internalization of fluorescent ligands with cell-surface receptors on cells and membranes. The time resolution available in these methods is sufficient to permit a detailed analysis of complex processes involved in cell activation, particularly receptor-G protein dynamics. A description of the kinetics and thermodynamics of these processes will contribute to our understanding of the basis of stimulus potency and efficacy. 

One common approach in the study of ligand binding to their receptors is to monitor fluorescence changes not directly but via energy transfer . The principles of this method are illustrated in O Figure 5-3. 

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