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Organization and Dynamics of NBD-Labeled Lipids in Membranes by Fluorescence recovery after photobleaching (FRAP) (CAT#: STEM-MT-0024-WXH)

Introduction

Lateral diffusion of membrane constituents plays an important role in membrane organization and represents a central theme in current models describing the structure and function of biological membranes. Fluorescence recovery after photobleaching (FRAP) is a widely used approach that provides information regarding dynamic properties and spatial distribution of membrane constituents.
On the basis of the unique concentration-dependent fluorescence emission properties of a fluorescently labeled cholesterol analogue modified at the tail region, 25-[N-[(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-methyl]amino]-27-norcholesterol (25-NBD-cholesterol), it exhibits local organization even at very low concentrations in membranes.

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Principle Applications Procedure Materials

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.

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