Elsevier

Methods

Volume 39, Issue 2, June 2006, Pages 147-153
Methods

Methods to measure the lateral diffusion of membrane lipids and proteins

https://doi.org/10.1016/j.ymeth.2006.05.008Get rights and content

Abstract

In this chapter, we discuss methods to measure lateral mobility of membrane lipids and proteins using techniques based on the light microscope. These methods typically sample lateral mobility in very small, micron-sized regions of the membrane so that they can be used to measure diffusion in regions of single cells. The methods are based on fluorescence from the molecules of interest or from light scattered from particles attached to single or small groups of membrane lipids or proteins. Fluorescence recovery after photobleaching (FRAP), fluorescence correlation spectroscopy (FCS) and Single particle tracking (SPT) are presented in that order. FRAP and FCS methodologies are described for a dedicated wide field microscope although many confocal microscopes now have software permitting these measurement to be made; nevertheless, the principles of the measurement are the same for a wide field or confocal microscope. SPT can be applied to trace the movements of single fluorescent molecules in membranes but this aspect will not be treated in detail.

Section snippets

Fluorescence recovery after photobleaching (FRAP)

Fluorescence recovery after photobleaching (FRAP) is a single cell technique used to study the mobility of fluorescent molecules. The method has been employed since the mid 1970s to examine molecular mobility in the plasma membrane, other organellar membranes and the cytoplasm [1]. A resurgence in use occurred in the 90s due to the advent of GFP fusion proteins [2].

In a FRAP experiment, a pulse of high intensity light from a laser is used to photobleach fluorophores, typically in a small

Fluorescence correlation spectroscopy (FCS)

Fluorescence correlation spectroscopy (FCS) is another technique which can be used to study translational mobilities in membranes. Similar to FRAP, this method requires molecules of interest to either be fluorescent or, alternatively, be conjugated to a fluorescent dye or protein. In this method, the fluctuations in the fluorescence intensity from a minute sample observation volume are recorded and temporally autocorrelated to reveal information about the concentration and dynamics of the

Single particle tracking (SPT)

Single particle tracking (SPT) is a powerful method for studying the movement of individual or small groups of proteins or lipids in the plasma membrane of live cells or in model membranes. The dynamic behavior of these molecules can be recorded and then analyzed to reveal the microstructure of the plasma membrane. By attaching antibody-coated sub-micron colloidal gold particles to molecules on the cell membrane, intense Rayleigh scattering can be detected from particles as small as 30 nm in

Acknowledgment

This work was supported by NIH GM 41402.

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