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Fluorescent labelling is the process of covalently attaching a fluorophore to another molecule, such as a protein or nucleic acid. This is generally accomplished using a reactive derivative of the fluorophore that selectively binds to a functional group contained in the target molecule. 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.
Fluorescent labels are generally used for detection of a protein or other labeled molecule via a fluorescence microscope, flow cytometer or some other fluorescence reading instrument. These can be useful in localization of a target within a cell, flow cytometry (FACS) analysis, western blot assays, and other immunoanalytical methods.
Fluorescent labelling is accomplished using a chemically reactive derivative of a fluorophore. Common reactive groups include:
- Isothiocyanate derivatives such as FITC and TRITC (derivatives of fluorescein and rhodamine) are reactive towards primary amines to form a thioureido linkage between the compound of interest and the dye.
- Succinimidyl esters such as NHS-fluorescein are reactive towards amino groups to form an amido bond.
- Maleimide activated fluorophores such as fluorescein-5-maleimide readily react with sulfhydryl groups. The sulfhydryl group adds to the double bond of the maleimide.
- In oligonucleotide synthesis, several phosphoramidite reagents containing protected fluorescein and other fluorophores, e.g. 6-FAM phosphoramidite 2, are reacted with hydroxy groups to allow the preparation of fluorophore-labelled oligonucleotides.
Reaction of any of these reactive dyes with another molecule results in a stable covalent bond formed between a fluorophore and a labelled molecule.
Following a fluorescent labelling reaction, it is often necessary to remove any nonreacted fluorophore from the labelled target molecule. This is often accomplished by size exclusion chromatography, taking advantage of the size difference between fluorophore and labelled protein, nucleic acid, etc. Fluorophores may interact with the separation matrix and reduce the efficiency of separation. For this reason, specialized dye removal columns that account for the hydrophobic properties of fluorescent dyes are sometimes used.
Reactive fluorescent dyes are available from many sources (see below). They can be obtained with different reactive groups for attachment to various functional groups within the target molecule. They are also available in labelling kits that contain all the components to carry out a labelling reaction.
Common fluorescent dyes
- Alexa Fluors
- Dylight fluors
- ATTO Dyes
- BODIPY Dyes
- SETA Dyes
- SeTau Dyes
- Presentation on Fluorescent labelling of biomolecules with organic probes | PharmaXChange.info
- Brush, C. K. Fluorescein Labelled Phosphoramidites. US Patent 5,583,236.