Study of Membrane Mobility and Microdomain Association of the Dopamine Transporter by Fluorescence recovery after photobleaching (FRAP) (CAT#: STEM-MT-0010-WXH)

Introduction

The dopamine transporter (DAT) controls the spatial and temporal dynamics of dopamine (DA) neurotransmission by driving reuptake of extracellular transmitter into presynaptic neurons.<br />Similar to other membrane proteins, the biogenic amine transporters are predicted not to be randomly distributed in the plasma membrane but associated with intracellular protein networks and plasma membrane microdomains that ensure appropriate spatial and temporal regulation of transporter function in the presynaptic membrane.

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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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