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In biochemistry, biotinylation is the process of covalently attaching a biotin tag to a molecule or surface. Biochemistry is the study of the chemical processes and transformations in living organisms. ...
Vitamin H redirects here. ...
Purpose
Determining extent of biotinylation Reaction conditions for biotinylation are chosen such that the target molecule (e.g. an antibody) is labelled with enough biotin substituents to purify or detect the molecule, but not so much that the biotin interferes with the function of the molecule. The HABA dye(2-(4-hydroxyazobenzene) benzoic acid) method is used to determine that extent of biotinylation. HABA dye is bound to avidin and yields a characteristic absorbance. When biotin, in the form of biotinylated protein or other molecule, is introduced, it displaces the dye, resulting in a change in absorbance at 500 nm. The absorbance change is directly proportional to the level of biotin in the sample.
Purification The biotin tag can be used in affinity chromatography together with a column that has avidin or streptavidin bound to it, which is the natural chelator for biotin. Affinity chromatography is a biochemical separation method that combines size fractionation capability of gel permeation chromatography with the ability to design a stationary phase that reversibly binds to a known subset of molecules. ...
Avidin is a protein found in egg white that binds with biotin, found in egg yolk. ...
Streptavidin (less commonly spelled streptavidine) is a tetrameric protein purified from Streptomyces avidinii that binds very tightly to the vitamin biotin with a dissociation constant (Kd) of ~ 10^(–15) M. This is one of the strongest biochemical interactions known, and is widely taken advantage of in scientific laboratories. ...
Chelation (from Greek, claw like) describes the reversible binding of an organic ligand, the chelator or chelating agent, to a metal ion, forming a metal complex, the chelate. ...
Detection This tag can also be used in detection of the protein via anti-biotin antibodies or avidine/streptavidin tagged detectors like horseradish peroxidase or a fluorescent dye. This can be useful in localization, ELISA assays, ELISPOT assays, western blots and other immunoanalytical methods. Each antibody binds to a specific antigen; an interaction similar to a lock and key. ...
The enzyme horseradish peroxidase, found in horseradish, is used extensively in molecular biology in antibody amplification and detection, among other things[1]. For example, In recent years the technique of marking neurons with the enzyme horseradish peroxidase (HRP) has become a major tool. ...
A fluorophore is a component of a molecule which causes a molecule to be fluorescent. ...
Elisa (born Elisa Toffoli on 19 December 1977) is an Italian singer and solo artist, writing and performing within several genres, notably rock, blues, soul and ambient. ...
The Enzyme-linked immunosorbent spot (ELISPOT) is a common method for monitoring immune responses in humans and animals. ...
Figure 1. ...
Other uses The non-covalent bond formed between biotin and avidin or streptavidin has a binding affinity that is tighter than most antigen and antibody bonds and approaches the strength of a covalent bond. This very tight binding makes labeling proteins with biotin a useful tool for applications such as affinity chromatography using immobilized avidin or streptavidin to separate the biotinylated protein from a mixture of other proteins and biochemicals. Biotinylated protein such as biotinylated bovine serum albumin (BSA) is used in solid-phase assays as a coating on the well surface in multiwell assay plates. Biotinylation of red blood cells has been used as a means of determining total blood volume without the use of radiolabels such as chromium 51, allowing volume determinations in low birth weight infants and pregnant women who could not otherwise be exposed to the required doses of radioactivity. Noncovalent bonding refers to a variety of interactions, that are not covalent in nature, between molecules or parts of molecules that provide force to hold the molecules or parts of molecules together usually in a specific orientation or conformation. ...
Covalent bonding is a form of chemical bonding that is characterized by the sharing of pairs of electrons between atoms, or sometimes between atoms and other covalent bonds. ...
Affinity chromatography is a biochemical separation method that combines size fractionation capability of gel permeation chromatography with the ability to design a stationary phase that reversibly binds to a known subset of molecules. ...
Human red blood cells Red blood cells are the most common type of blood cell and are the vertebrate bodys principal means of delivering oxygen to body tissues via the blood. ...
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