Methods to measure the lateral diffusion of membrane lipids and proteins
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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