Analysis of connexin36 gap junction coupling by Fluorescence recovery after photobleaching (FRAP) (CAT#: STEM-MT-0027-WXH)

Introduction

Gsp junction (GJ) channels consist of two apposed hemichannels from contiguous cells. In vertebrates, each hemichannel is formed by six protein subunits of the connexin (Cx) family.
In humans, 21 different Cx isoforms have been identified. These isoforms are differentially expressed in various tissues and exhibit different biophysical and biochemical properties. Among the Cx family, Cx36 is mainly expressed in the adult mammalian central nervous system, where it forms electrical synapses. It has been shown that Cx36-containing electrical synapses play an important role in facilitating synchronous or phase-locked activity of neuronal networks, which underlie a variety of cognitive processes. Cx36 also forms GJs between pancreatic beta cells, where it plays an important role in insulin secretion and glycaemic control.

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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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