Diffusion and Binding Properties Investigated by Fluorescence Correlation Spectroscopy (FCS)

Abstract

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.

Peptide Nanoparticles Serve as a Powerful Platform for the Immunogenic Display of Poorly Antigenic Actin Determinants

“…A number of recent studies have used fluorescence correlation spectroscopy to measure antigen-antibody interactions. [36][37][38] The combination with a confocal microscope will not only improve signalto-noise ratios in spectroscopic measurements, but also will allow simultaneous confocal imaging of the labeled specimen. Confocal images (Fig.…”

Measurement of diffusion in articular cartilage using fluorescence correlation spectroscopy

“…Further practical considerations in the calculation of FCS curves from a fluorescence intensity trace are detailed elsewhere [35-37]. …”

DNA oligomer binding in competition exhibits cooperativity

“…the focal volume of the objective (40× C-Apochromat, ZEISS, Germany). We deduce the binding constant from changes in diffusion time of the labeled strand as a function of the concentration of its unlabeled binding partner [68]. The characteristic diffusion times for a free, labeled strand D 1 t and a bound strand D 2 t are determined first.…”

“…The fraction of duplex conformations q as well as N are free fitting parameters. The binding constant is determined from the concentration dependency of q [68,69]. Figure 1 shows the measured fluorescence autocorrelation functions for the single (circles, q = 0) and bound strands (diamonds, q = 1) in P-PM hybridization.…”