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Measurement of lateral diffusion in phospholipid multibilayers by Fluorescence recovery after photobleaching (FRAP) (CAT#: STEM-MT-0003-WXH)

Introduction

Movement of lipids within each leaflet of the lipid bilayer occurs readily and rapidly due to membrane fluidity. This type of movement is called lateral diffusion and can be measured by the technique called FRAP. <br />Membrane protein lateral diffusion can be constrained in several ways: Diffusion can be slower than that predicted for a simple, fluid lipid bilayer; diffusion can be confined to certain regions within the total membrane; and diffusion may not be equally probable in all directions, i.e. it may be anisotropic.




Principle

Fluorescence recovery after photobleaching (FRAP) is a microscopy technique capable of quantifying the mobility of molecules within cells. By exploiting the phenomenon of photobleaching, fluorescent mole- cules within a region of interest can be selectively and irreversibly 'turned off'. It is capable of quantifying the two-dimensional lateral diffusion of a molecularly thin film containing fluorescently labeled probes, or to examine single cells.

Applications

• Characterization of the mobility of individual lipid molecules within a cell membrane.
• Analysis of molecule diffusion within the cell
• Study of protein interaction partners, organelle continuity and protein trafficking.

Procedure

1. An initial fluorescence of fluorescent molecules is measured in the region of interest (ROI).
2. The fluorescent molecules are rapidly photobleached by focusing the high-intensity laser beam onto the defined area.
3. The exchange of bleached molecules with unbleached molecules from the surrounding region is followed over time using a low-intensity laser.

Materials

• Optical microscope.
• Light source.
• Fluorescent probe.