Cellular Biology

Molecular Imaging Fluorescent Protein Tags Explained

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Proteins tagged with fluorescent markers are extremely versatile tools in cell and molecular biology research. A fluorescent marker is, as the name suggests, a molecule that emits light of a particular wavelength following exposure to a photons of a shorter wavelength, for example a laser beam. If a fluorescent marker is a reasonable size and chemically stable enough to be attached to a protein, it is potentially useful to molecular biologists.

In many cases, the proteins being tagged are monoclonal antibodies generated against a particular target of interest such as a cell surface receptor, a cytoskeletal protein, or a cellular organelle.  In some cases, a fluorescent label is used to track the fate of a particular protein as it is processed in the ER and Golgi complex, transported along microtubules, or secreted from the cell altogether. In still other cases, two or more fluorescently tagged proteins are introduced into the same cell to see whether they localize in the same cellular region or in separate organelles.

One of the first techniques that made widespread use of fluorescent tagged proteins was flow cytometry or FACS (Fluorescence Activated Cell Sorting). In this method, cells are incubated with antibodies conjugated with fluorescent dyes such as FITC (fluoroscein isothiocyanate), which emits green wavelengths of light; PE (phycoerythrin), or Texas Red, both of which emit red wavelengths of light. Newer fluorescent dyes emit yellow and blue wavelengths as well.

Following a 30 to 60 minute incubation, the cells are suspended in solution and aspirated into the flow cytometer. Once inside, the cells are channeled into a single file line and exposed to one or more laser beams. The machine then records the light signals emitted from the fluorescent antibodies attached to the cells. Depending upon how many wavelengths the machine can detect, six or more different cell surface receptors can be analyzed at once. Other fluorescence imaging techniques that have gained popularity over the past two decades include immunohistochemistry and fluorescence microscopy.

In addition to fluorescent dyes, fluorescent proteins (the entire protein acts as the marker) have come into widespread use in the last several years. Transgenic organisms expressing GFP (Green Fluorescent Protein) revolutionized the field of developmental biology starting in the 1990's. Simply by injecting the GFP gene into embryonic cells, scientists could now track the developmental fate of individual cells in organisms ranging from worms like C. elegans to vertebrates like zebrafish. Red and yellow fluorescent proteins (RFP and YFP) were developed shortly afterward; both are commonly used to visualize transgenic cells in vitro and in vivo.

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