Diffusion and binding properties investigated by Fluorescence Correlation Spectroscopy (FCS)
Department of Biochemistry and Molecular Pharmacology; RNA Therapeutics Institute
Medical Subject Headings
*Algorithms; Binding Sites; Biopolymers; Diffusion; Fluorescent Antibody Technique; Kinetics; Protein Interaction Mapping; Spectrometry, Fluorescence; Statistics as Topic
During the last years, Fluorescence Correlation Spectroscopy (FCS) has proven to be a powerful tool for basic research in many applications. The combination of a minimal detection volume in the femtoliter range coupled with very high sensitivity extends the possibilities to design sensitive homogeneous tests. In this article we illustrate the analysis of binding processes with FCS based on the changes in diffusion characteristics of GFP upon binding to an antibody. Problems induced by highly heterogeneous samples are discussed and differences of GFP binding to a monoclonal and a polyclonal antibody are shown and analyzed. We stress data processing, limitations and useful approximations in FCS methodology. Basic ideas of data acquisition and processing as well as new developments and applications are presented.
fluorescence correlation spectroscopy, gfp, kinetics, binding assay, antibody
Grunwald, David; Cardoso, M. C.; Leonhardt, Heinrich; and Buschmann, Volker, "Diffusion and binding properties investigated by Fluorescence Correlation Spectroscopy (FCS)" (2005). Biochemistry and Molecular Pharmacology Publications and Presentations. Paper 178.