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Study of RNA Motion by Fluorescence recovery after photobleaching (FRAP) (CAT#: STEM-MT-0014-WXH)

Introduction

Eukaryotic cells contain a myriad of RNA species that are implicated in virtually all aspects of gene expression. Inside the living cell, RNA molecules move between subcellular compartments and assemble into distinct macromolecular complexes that are highly dynamic over time and space. A variety of time-lapse microscopy techniques are currently available to track these movements using fluorescent tags that bind to the RNA.




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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