Equilibrium, Kinetics, Diffusion and Self‐Association of Proteins at Membrane Surfaces: Measurement by Total Internal Reflection Fluorescence Microscopy

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Abstract
The equilibrium, kinetics, diffusion and self-association of proteins at membrane/solution interfaces may deviate substantially from these processes in bulk solution. A set of methods for examining these phenomena combines substrate-supported planar model membranes and the use of evanescent illumination with laser-based, quantitative fluorescence microscopy. Measurement of the steady-state, surface-associated fluorescence can be used to examine the thermodynamic properties of proteins at membranes. When combined with fluorescence photobleaching recovery, this technique provides information about membrane-binding kinetics; and when combined with fluorescence pattern photobleaching recovery, measurement of the translational diffusion coefficients of proteins weakly bound to membranes is possible. The use of polarized evanescent illumination can provide information about the orientation distributions of adsorbed fluorophores. Fluorescence correlation spectroscopy provides information about the self-association (e.g. dimerization) of membrane-associated proteins.
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