Measurement of Diffusion in cells by Fluorescence recovery after photobleaching (FRAP) (CAT#: STEM-MT-0029-WXH)

Introduction

Cell diffusion is a type of passive transport across the cell membrane. Therefore, it does not require energy. Diffusion relies on the basic principle that molecules will tend to reach equilibrium and will therefore move from a region of high concentration to a region of low concentration.
In other words, diffusion is the type of cellular transport where molecules freely flow from the side of the membrane where the concentration is high to the side where it is low.

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Principle Applications Procedure Materials Notes Related Products

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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