The green fluorescent protein (GFP) is a protein that exhibits green fluorescence when exposed to light in the blue to ultraviolet range. The label GFP traditionally refers to the protein first isolated from the jellyfish Aequorea victoria and is sometimes called avGFP. However, GFPs have been found in other organisms including corals, sea anemones, zoanithids, copepods and lancelets.
In molecular biology and biotechnology, a fluorescent tag, also known as a fluorescent label or fluorescent probe, is a molecule that is attached chemically to aid in the detection of a biomolecule such as a protein, antibody, or amino acid. Generally, fluorescent tagging, or labeling, uses a reactive derivative of a fluorescent molecule known as a fluorophore. The fluorophore selectively binds to a specific region or functional group on the target molecule and can be attached chemically or biologically. Various labeling techniques such as enzymatic labeling, protein labeling, and genetic labeling are widely utilized. Ethidium bromide, fluorescein and green fluorescent protein are common tags. The most commonly labelled molecules are antibodies, proteins, amino acids and peptides which are then used as specific probes for detection of a particular target.
A fluorophore is a fluorescent chemical compound that can re-emit light upon light excitation. Fluorophores typically contain several combined aromatic groups, or planar or cyclic molecules with several π bonds.
Förster resonance energy transfer (FRET), fluorescence resonance energy transfer, resonance energy transfer (RET) or electronic energy transfer (EET) is a mechanism describing energy transfer between two light-sensitive molecules (chromophores). A donor chromophore, initially in its electronic excited state, may transfer energy to an acceptor chromophore through nonradiative dipole–dipole coupling. The efficiency of this energy transfer is inversely proportional to the sixth power of the distance between donor and acceptor, making FRET extremely sensitive to small changes in distance.
Protein tags are peptide sequences genetically grafted onto a recombinant protein. Tags are attached to proteins for various purposes. They can be added to either end of the target protein, so they are either C-terminus or N-terminus specific or are both C-terminus and N-terminus specific. Some tags are also inserted at sites within the protein of interest; they are known as internal tags.
Bimolecular fluorescence complementation is a technology typically used to validate protein interactions. It is based on the association of fluorescent protein fragments that are attached to components of the same macromolecular complex. Proteins that are postulated to interact are fused to unfolded complementary fragments of a fluorescent reporter protein and expressed in live cells. Interaction of these proteins will bring the fluorescent fragments within proximity, allowing the reporter protein to reform in its native three-dimensional structure and emit its fluorescent signal. This fluorescent signal can be detected and located within the cell using an inverted fluorescence microscope that allows imaging of fluorescence in cells. In addition, the intensity of the fluorescence emitted is proportional to the strength of the interaction, with stronger levels of fluorescence indicating close or direct interactions and lower fluorescence levels suggesting interaction within a complex. Therefore, through the visualisation and analysis of the intensity and distribution of fluorescence in these cells, one can identify both the location and interaction partners of proteins of interest.
Within the field of molecular biology, a protein-fragment complementation assay, or PCA, is a method for the identification and quantification of protein–protein interactions. In the PCA, the proteins of interest are each covalently linked to fragments of a third protein. Interaction between the bait and the prey proteins brings the fragments of the reporter protein in close proximity to allow them to form a functional reporter protein whose activity can be measured. This principle can be applied to many different reporter proteins and is also the basis for the yeast two-hybrid system, an archetypical PCA assay.
Kaede is a photoactivatable fluorescent protein naturally originated from a stony coral, Trachyphyllia geoffroyi. Its name means "maple" in Japanese. With the irradiation of ultraviolet light (350–400 nm), Kaede undergoes irreversible photoconversion from green fluorescence to red fluorescence.
EosFP is a photoactivatable green to red fluorescent protein. Its green fluorescence (516 nm) switches to red (581 nm) upon UV irradiation of ~390 nm due to a photo-induced modification resulting from a break in the peptide backbone near the chromophore. Eos was first discovered as a tetrameric protein in the stony coral Lobophyllia hemprichii. Like other fluorescent proteins, Eos allows for applications such as the tracking of fusion proteins, multicolour labelling and tracking of cell movement. Several variants of Eos have been engineered for use in specific study systems including mEos2, mEos4 and CaMPARI.
Jennifer Lippincott-Schwartz is a Senior Group Leader at Howard Hughes Medical Institute's Janelia Research Campus and a founding member of the Neuronal Cell Biology Program at Janelia. Previously, she was the Chief of the Section on Organelle Biology in the Cell Biology and Metabolism Program, in the Division of Intramural Research in the Eunice Kennedy Shriver National Institute of Child Health and Human Development at the National Institutes of Health from 1993 to 2016. Lippincott-Schwartz received her PhD from Johns Hopkins University, and performed post-doctoral training with Richard Klausner at the NICHD, NIH in Bethesda, Maryland.
mCherry is a member of the mFruits family of monomeric red fluorescent proteins (mRFPs). As an RFP, mCherry was derived from DsRed of Discosoma sea anemones, unlike green fluorescent proteins (GFPs) which are often derived from Aequorea victoria jellyfish. Fluorescent proteins are used to tag components in cells so that they can be studied using fluorescence spectroscopy and fluorescence microscopy. mCherry absorbs light between 540 and 590 nm and emits light in the range of 550-650 nm. mCherry belongs to the group of fluorescent protein chromophores used as instruments to visualize genes and analyze their functions in experiments. Genome editing has been improved greatly through the precise insertion of these fluorescent protein tags into the genetic material of many diverse organisms. Most comparisons between the brightness and photostability of different fluorescent proteins have been made in vitro, removed from biological variables that affect protein performance in cells or organisms. It is hard to perfectly simulate cellular environments in vitro, and the difference in environment could have an effect on the brightness and photostability.
Chemically induced dimerization (CID) is a biological mechanism in which two proteins bind only in the presence of a certain small molecule, enzyme or other dimerizing agent. Genetically engineered CID systems are used in biological research to control protein localization, to manipulate signalling pathways and to induce protein activation.
SNAP-tag® is a self-labeling protein tag commercially available in various expression vectors. SNAP-tag is a 182 residue polypeptide that can be fused to any protein of interest and further specifically and covalently tagged with a suitable ligand, such as a fluorescent dye. Since its introduction, SNAP-tag has found numerous applications in biochemistry and for the investigation of the function and localisation of proteins and enzymes in living cells.
Epicocconone is a long Stokes' shift fluorogenic natural product found in the fungus Epicoccum nigrum. Though weakly fluorescent in water it reacts covalently yet reversibly with primary amines such as those in proteins to yield a product with a strong orange-red emission (610 nm). Epicoconone is notable because it the first covalent/reversible/turn-on fluorophore to be discovered and is a natural product with a new fluorescent scaffold. It is also cell membrane permeable, unlike many other fluorophores, and subsequently can be used in in vivo applications. Additionally, this dye can be used as a sensitive total protein stain for 1D and 2D electrophoresis, quantitative determination of protein concentration, making it a powerful loading control for Western blots.
Calcium imaging is a microscopy technique to optically measure the calcium (Ca2+) status of an isolated cell, tissue or medium. Calcium imaging takes advantage of calcium indicators, fluorescent molecules that respond to the binding of Ca2+ ions by fluorescence properties. Two main classes of calcium indicators exist: chemical indicators and genetically encoded calcium indicators (GECI). This technique has allowed studies of calcium signalling in a wide variety of cell types. In neurons, action potential generation is always accompanied by rapid influx of Ca2+ ions. Thus, calcium imaging can be used to monitor the electrical activity in hundreds of neurons in cell culture or in living animals, which has made it possible to observe the activity of neuronal circuits during ongoing behavior.
A FMN-binding fluorescent protein (FbFP), also known as a LOV-based fluorescent protein, is a small, oxygen-independent fluorescent protein that binds flavin mononucleotide (FMN) as a chromophore.
Small ultra red fluorescent protein (smURFP) is a class of far-red fluorescent protein evolved from a cyanobacterial phycobiliprotein, α-allophycocyanin. Native α-allophycocyanin requires an exogenous protein, known as a lyase, to attach the chromophore, phycocyanobilin. Phycocyanobilin is not present in mammalian cells. smURFP was evolved to covalently attach phycocyanobilin without a lyase and fluoresce, covalently attach biliverdin and fluoresce, blue-shift fluorescence to match the organic fluorophore, Cy5, and not inhibit E. coli growth. smURFP was found after 12 rounds of random mutagenesis and manually screening 10,000,000 bacterial colonies.
Super-resolution dipole orientation mapping (SDOM) is a form of fluorescence polarization microscopy (FPM) that achieved super resolution through polarization demodulation. It was first described by Karl Zhanghao and others in 2016. Fluorescence polarization (FP) is related to the dipole orientation of chromophores, making fluorescence polarization microscopy possible to reveal structures and functions of tagged cellular organelles and biological macromolecules. In addition to fluorescence intensity, wavelength, and lifetime, the fourth dimension of fluorescence—polarization—can also provide intensity modulation without the restriction to specific fluorophores; its investigation in super-resolution microscopy is still in its infancy.
Red fluorescent protein (RFP) is a fluorophore that fluoresces red-orange when excited. Several variants have been developed using directed mutagenesis. The original was isolated from Discosoma, and named DsRed. Others are now available that fluoresce orange, red, and far-red.
In molecular biology, the PYP domain is a p-coumaric acid-binding protein domain. They are present in various proteins in bacteria.
