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Fluorescent Protein Plasmid Resource: Subcellular Localization, FRET, Biosensors, and more

Posted in: Protein Expression and Analysis

Fluorescent molecules are a class of molecules capable of becoming excited (absorbing) light at specific wavelengths then emitting that light as a longer wavelength after some interval of time. Since the discovery of the original green fluorescent protein (GFP) in the 1960s, researchers have been improving and modifying fluorescent proteins to yield brighter, more photostable fluores capable of emitting light across the entire color spectrum in a variety of temperature and pH settings.

Addgene, a non-profit plasmid repository, is working with hundreds of labs around the world to assemble a collection of fluorescent protein tools for a variety of in-vitro and in-vivo applications.

Create your Fusion Protein

To understand the localization and/or function of your protein of interest, you may want to fuse it to a fluorescent protein. You will then be able to visualize where your protein is in live cells, or even in live animals, using fluorescence microscopy.

Find excitation/emission wavelengths for common fluorescent proteins, then obtain the appropriate plasmid for cloning with Addgene’s Fluorescent Protein Backbone Resource.

FRET for measuring protein interactions

Förster resonance energy transfer, or FRET, is a process by which energy is transferred from an excited donor fluorophore to an acceptor molecule. The efficiency of the energy transfer is measured using fluorescence microscopy by exciting the donor and measuring the emission of the acceptor.

FRET is a powerful tool for studying many biological processes including conformation changes within proteins as well as protein-protein interactions. Create your own FRET pairs and find FRET standards in Addgene’s FRET Guide.

Biosensors with fluorescent readouts

Genetically encoded fluorescent biosensors are fluorescent proteins attached to an additional protein sequence that make it sensitive to small biomolecules or other physiological intracellular processes. Fluorescent biosensors are introduced into cells, tissues or organisms to allow for detection by fluorescence microscopy as a difference in FRET efficiency, translocation of the fluorescent protein or modulation of the fluorescent properties of a single fluorescent protein. Benefits of fluorescent biosensors include photostability, which allows for long-term imaging, and the flexibility to design custom biosensors specific for targeting cellular compartments and organelles. In addition, biosensors permit investigation of a signaling pathway or measurement of a biomolecule while largely preserving spatial and temporal cellular processes.

Addgene has a library of plasmids that encode fluorescent biosensors and kits for constructing custom biosensors. Learn more by visiting Addgene’s Biosensor collection.

Optogenetics

Originally developed in 2006, optogenetics research utilizes light to detect, measure, and control molecular signals, cells, and groups of cells in living organisms and tissue to understand activity and the effects of alterations to this activity.

These genetically-encoded tools come in two primary categories: actuators and sensors. Optogenetic actuators are used for light-activated control of neurons (for example, microbial opsins). Optogenetic sensors monitor and measure fluctuations in molecular signals, such as calcium, glutamate, pH, and voltage.

To find plasmids for optogenetics by category and application, visit Addgene’s Optogenetics Resource.

Subcellular Localization

The subcellular localization of a protein is often tied to its function, so it’s important to determine where your protein of interest resides. Addgene has assembled a collection of fluorescent fusion proteins that are targeted to various organelles or subcellular structures. You can use these tools to assess whether your protein is targeted to the same structures as these well-characterized proteins.

Determine what subcellular structure your protein is associated with using Addgene’s Subcellular Localization Guide.

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