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S. cerevisiae septins revealed with fluorescent microscopy utilizing fluorescent labeling. 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.
In these embryos the concentration of Bicoid protein could be accurately measured either by the intensity of the autofluorescence of the GFP protein itself (not shown) or by the intensity of fluorescence of an anti-GFP antibody tagged with a fluorescent dye, as shown in a surface view of the embryo here.
SNAP-tag reaction scheme. SNAP-tag® is a self-labeling protein tag commercially available in various expression vectors. SNAP-tag is a 182 residue polypeptide (19.4 kDa) that can be fused to any protein of interest and further specifically and covalently tagged with a suitable ligand, such as a fluorescent dye.
More generally they are covalently bonded to macromolecules, serving as a markers (or dyes, or tags, or reporters) for affine or bioactive reagents (antibodies, peptides, nucleic acids). Fluorophores are notably used to stain tissues, cells, or materials in a variety of analytical methods, such as fluorescent imaging and spectroscopy .
Cyanine dyes are used to label proteins, antibodies, peptides, nucleic acid probes, and any kind of other biomolecules to be used in a variety of fluorescence detection techniques: flow cytometry, microscopy (mainly the visible range, but also UV and IR), microplate assays, microarrays, as well as "light-up probes," and in vivo imaging.
Not fluorescent by itself, it can bind selectively a fluorogenic chromophore derived from 4-hydroxybenzylidene rhodanine, which is itself non fluorescent unless bound. Once bound, the pair of molecules goes through a unique fluorogen activation mechanism based on two spectroscopic changes, increase of fluorescence quantum yield and absorption ...
A simplified Jablonski diagram illustrating the change of energy levels.. The principle behind fluorescence is that the fluorescent moiety contains electrons which can absorb a photon and briefly enter an excited state before either dispersing the energy non-radiatively or emitting it as a photon, but with a lower energy, i.e., at a longer wavelength (wavelength and energy are inversely ...
In the case of synthetic peptides, the advantage is the amino acid sequence is easily accessible, but the peptides do not always resemble the 3-D structure or post-translational modification found in the native form of the protein. Therefore, antibodies that are produced to work against a synthetic peptide may have problems with the native 3-D ...
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