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Study of Molecular diffusion in cheese by Fluorescence recovery after photobleaching (FRAP) (CAT#: STEM-MT-0056-WXH)

Introduction

Molecular diffusion, as characterized by diffusion coefficients, is a key feature in the manufacture of certain food products, especially those from the dairy industry. During the manufacture of cheese, the coagulation of caseins forms a protein gel structure that entraps the aqueous phase of milk (whey proteins, minerals…), fat and micro-organisms. Cheese ripening involves complex microbiological and biochemical reactions that take place within this protein gel resulting in the development of flavor and texture characteristics, specific to each cheese variety. Such protein gel structure (also referred to as a protein network) acts like a sieve, where the gel strands impedes the diffusing molecules; a denser gel structure leading to a greater obstruction.




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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