Biotinylation
In biochemistry, biotinylation is the process of covalently attaching a biotin tag to a molecule or surface.
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 (also streptavidin or Neutravidin) bound to it, which is the natural chelator for biotin.
Detection
This tag can also be used in detection of the protein via anti-biotin antibodies or avidin/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.
Other uses
The non-covalent bond formed between biotin and avidin or streptavidin has a binding affinity that is higher 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.