Fluorescent labelling
Encyclopedia
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.
, 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.
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 effiency 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.
Fluorophore
A fluorophore, in analogy to a chromophore, is a component of a molecule which causes a molecule to be fluorescent. It is a functional group in a molecule which will absorb energy of a specific wavelength and re-emit energy at a different wavelength...
to another molecule, such as a protein
Protein
Proteins are biochemical compounds consisting of one or more polypeptides typically folded into a globular or fibrous form, facilitating a biological function. A polypeptide is a single linear polymer chain of amino acids bonded together by peptide bonds between the carboxyl and amino groups of...
or nucleic acid
Nucleic acid
Nucleic acids are biological molecules essential for life, and include DNA and RNA . Together with proteins, nucleic acids make up the most important macromolecules; each is found in abundance in all living things, where they function in encoding, transmitting and expressing genetic information...
. This is generally accomplished using a reactive derivative of the fluorophore that selectively binds to a functional group
Functional group
In organic chemistry, functional groups are specific groups of atoms within molecules that are responsible for the characteristic chemical reactions of those molecules. The same functional group will undergo the same or similar chemical reaction regardless of the size of the molecule it is a part of...
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.
Detection
Fluorescent labels are generally used for detection of a protein or other labelled molecule via a fluorescence microscopeFluorescence microscope
A fluorescence microscope is an optical microscope used to study properties of organic or inorganic substances using the phenomena of fluorescence and phosphorescence instead of, or in addition to, reflection and absorption...
, flow cytometer or some other fluorescence reading instrument. These can be useful in localization of a target within a cell, flow cytometry
Flow cytometry
Flow cytometry is a technique for counting and examining microscopic particles, such as cells and chromosomes, by suspending them in a stream of fluid and passing them by an electronic detection apparatus. It allows simultaneous multiparametric analysis of the physical and/or chemical...
(FACS) analysis, western blot
Western blot
The western blot is a widely used analytical technique used to detect specific proteins in the given sample of tissue homogenate or extract. It uses gel electrophoresis to separate native proteins by 3-D structure or denatured proteins by the length of the polypeptide...
assays, and other immunoanalytical methods.
Labelling techniques
Fluorescent labelling is accomplished using a chemically reactive derivative of a fluorophore. Common reactive groups include:- IsothiocyanateIsothiocyanateIsothiocyanate is the chemical group –N=C=S, formed by substituting sulfur for oxygen in the isocyanate group. Many natural isothiocyanates from plants are produced by enzymatic conversion of metabolites called glucosinolates. These natural isothiocyanates, such as allyl isothiocyanate, are also...
derivatives such as FITCFluorescein isothiocyanateFluorescein isothiocyanate is a derivative of fluorescein used in wide-ranging applications including flow cytometry. FITC is the original fluorescein molecule functionalized with an isothiocyanate reactive group , replacing a hydrogen atom on the bottom ring of the structure...
and TRITC (derivatives of fluoresceinFluoresceinFluorescein is a synthetic organic compound available as a dark orange/red powder soluble in water and alcohol. It is widely used as a fluorescent tracer for many applications....
and rhodamineRhodamineRhodamine is a family of related chemical compounds, fluorone dyes. Examples are Rhodamine 6G and Rhodamine B. They are used as a dye and as a dye laser gain medium. They are often used as a tracer dye within water to determine the rate and direction of flow and transport...
) 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.
- MaleimideMaleimideMaleimide is the chemical compound with the formula H2C22NH . This unsaturated imide is an important building block in organic synthesis. The name is a contraction of maleic acid and imide, the -CNHC- functional group...
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 synthesisOligonucleotide synthesisOligonucleotide synthesis is the chemical synthesis of relatively short fragments of nucleic acids with defined chemical structure . The technique is extremely useful in current laboratory practice because it provides a rapid and inexpensive access to custom-made oligonucleotides of the desired...
, several phosphoramidite reagents containing protected fluorescein and other fluorophores, e.g. 6-FAM phosphoramiditeFluorescein amiditeFluorescein amidite, abbreviated as FAM , is an important synthetic equivalent of fluorescein dye used in oligonucleotide synthesis and molecular biology...
2, are reacted with hydroxy groups to allow the preparation of fluorophore-labelled oligonucleotideOligonucleotideAn oligonucleotide is a short nucleic acid polymer, typically with fifty or fewer bases. Although they can be formed by bond cleavage of longer segments, they are now more commonly synthesized, in a sequence-specific manner, from individual nucleoside phosphoramidites...
s.
Reaction of any of these reactive dyes with another molecule results in a stable covalent bond
Covalent bond
A covalent bond is a form of chemical bonding that is characterized by the sharing of pairs of electrons between atoms. The stable balance of attractive and repulsive forces between atoms when they share electrons is known as covalent bonding....
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
Size exclusion chromatography
Size-exclusion chromatography is a chromatographic method in which molecules in solution are separated by their size, and in some cases molecular weight . It is usually applied to large molecules or macromolecular complexes such as proteins and industrial polymers...
, taking advantage of the size difference between fluorophore and labelled protein, nucleic acid, etc. Fluorophores may interact with the separation matrix and reduce the effiency 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
- fluoresceinFluoresceinFluorescein is a synthetic organic compound available as a dark orange/red powder soluble in water and alcohol. It is widely used as a fluorescent tracer for many applications....
- rhodamineRhodamineRhodamine is a family of related chemical compounds, fluorone dyes. Examples are Rhodamine 6G and Rhodamine B. They are used as a dye and as a dye laser gain medium. They are often used as a tracer dye within water to determine the rate and direction of flow and transport...
- Alexa Fluors
- Dylight fluorsDyLight FluorThe DyLight Fluor family of fluorescent dyes are produced by Dyomics in collaboration with Thermo Fisher Scientific. DyLight dyes are typically used in biotechnology and research applications as biomolecule, cell and tissue labels for fluorescence microscopy, cell biology or molecular...
- ATTO DyesATTO dyesATTO Dyes are a series of fluorescent labels and dyes manufactured by ATTO-TEC GmbH in Siegen, Germany. The dyes have been designed for application in the area of life science, e.g...
- BODIPYBODIPYBODIPY, short for boron-dipyrromethene, is a class of fluorescent dyes. It is composed of dipyrromethene complexed with a disubstituted boron atom, typically a BF2 unit. The IUPAC name for the BODIPY core is 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene...
Dyes