Study of Mobility of Adsorbed Proteins by Fluorescence recovery after photobleaching (FRAP) (CAT#: STEM-MT-0048-WXH)

Introduction

Extensive research has been performed on the macroscopic scale to probe protein adsorption, particularly in the areas of adsorption kinetics, equilibrium, and relevant transport phenomena for different systems. However, information considering the molecular-level behavior of adsorbed proteins forms a relatively small fraction of this work, and understanding of such issues as lateral mobility, conformation, orientation, and ordering of protein molecules is thus incomplete. It is especially translational mobility of adsorbed protein that we investigate here, which is relevant to such applications as protein transport in chromatography and formation of structured adlayers at surfaces.

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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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