Study of Protein Surface Diffusion by Fluorescence recovery after photobleaching (FRAP) (CAT#: STEM-MT-0059-WXH)

Introduction

Lateral diffusion of proteins at interfaces is important for many biological systems. It leads to enhanced receptorligand recognition in the cell membrane and is relevant for understanding the localization of different membrane receptors.
It is evident that protein properties, e.g., conformational flexibility and molecular weight, combined with surface properties, e.g., hydrophobicity and charge, affect the diffusion rate, but the exact mechanism is not well explained.

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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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