Binding of proteins to specific target sites in membranes measured by total internal reflection fluorescence microscopy

Abstract

A new quantitative technique for measuring the binding of proteins to membranes is described. The method is based on a combination of total internal reflection fluorescence microscopy and the preparation of supported planar bilayers. Specific and reversible binding of a fluorescence-labeled monoclonal antibody to lipid haptens that were embedded in supported bilayers has been measured by this technique and compared to binding experiments that were conducted on membrane vesicles in solution. Equilibrium binding constants and kinetic parameters have been determined and used to expand the picture of the antibody-lipid hapten reaction. Estimates demonstrate that this technique is capable of measuring a broad range of binding constants (down to about 10(4) M-1) using only small amounts of ligand and receptor.

A Silicate Gel for Promoting Deposition of Lipid Bilayers

“…A number of surface-sensitive techniques have been used to study the interaction of vesicle suspensions with solid surfaces, including electrochemical (10) and optical (5,(11)(12)(13) methods. Recently, acoustic wave devices have also been applied to these studies and have been shown to be able to monitor the mass and viscoelastic changes occurring during vesicle fusion on the device surface (14)(15)(16).…”

Fluorescence

“…In some applications, an objective lens with a large numerical aperture acts as the medium with the higher refractive index, and also serves to collect fluorescence emitted by the sample. Excitation schemes utilizing total internal reflection have been used to measure binding of fluorescent ligands to protein adsorbed on a glass surface [205][206][207][208], and to probe the orientation and motions of molecules in planar lipid bilayers deposited on such a surface [164,165,209].…”

Ionophore/Lipid Bilayer Assembly on Soft Organic Electrodes for Potentiometric Detection of K+ Ions

“…The fluidity and stability of the solid-supported membranes planar surfaces have a distinct advantage over freestanding "black lipid" membranes or spherical lipid vesicles models because it makes it possible to carry out experiments and use analytical methods that are difficult or impossible to use with other model systems. Methods like total interference fluorescence, nuclear magnetic resonance (NMR), Fourier-transform infrared spectroscopy, Surface Plasmon resonance, and X-ray and neutron scattering can all be used to probe the structural and dynamic properties of solid supported membranes (Kalb 1992, Baruerl 1990, Tatulian 1995, Terretaz 1993, Kjaer 1987, Kalb 1990, Majeski 1998.…”