Analysis of Ligand Binding and Cross-Linking of Receptors in Solution and on Cell Surfaces

Fluorescence measurements are among the most powerful methods for investigating structure and structural changes on cell surfaces. The basic strength of this method is that nanomolar concentrations of fluorophores can be detected in the presence of a high level of background noise provided by the cells. An ideal fluorescent probe absorbs and emits at wavelengths not in common with the cellular components, has a high quantum yield, and can be placed specifically into a location such that it is sensitive to the structural aspect of interest. Recently there has been a great expansion in the commercial availability of fluorescent probes with a broad range of fluorescent properties and reactive groups for conjugation (e.g., Haugland, 1989). The requirement for specific placement on a macromolecular/cellular complex remains the most challenging experimentally. In this regard, specific ligands and specific monoclonal antibodies that can be fluorescently modified are valuable reagents. In this chapter we describe our use of quantitative fluorescence measurements to investigate the binding properties of cell surface receptors.