Biotinylation
| Eponymous biotin |
|---|
 Biotin |
The biotinylation refers to the process of covalent binding of biotin to a molecule , usually a protein or DNA . With some proteins this happens as a post-translational modification . Biotinylation is also used specifically in the laboratory to mark a molecule for the purpose of molecular marking (e.g. for detection in ELISA , ELISPOT , immunohistochemistry and Western blot ) or for purification . Then the strong specific interaction between biotin and avidin or streptavidin is used.
Application examples
Biotinylation can be artificially introduced into molecules, e.g. B. in vivo by protein tags such as Avi-Tag, BCCP-Tag, Strep-Tag or in vitro by coupling with reactive biotins such as NHS- Biotin.
Biotinylation can be used to purify biotin-labeled substances (e.g. nucleic acids ). Here, the biotin-labeled target substance is placed on an affinity chromatography column, the column material of which carries covalently bound avidin. The biotin binds the avidin, then the surface is rinsed so that unbound substances are washed away. The biotinylated molecules can then also be separated from the surface using suitable biotin-containing or denaturing elution solutions.
The coupling with biotin is also used to mark certain molecules (e.g. protein ). In some cases the molecules to be detected are directly biotinylated and can, for example, be made visible via an avidin-coupled dye. Often, however, a direct detection is dispensed with in favor of the signal strength and a number of intermediate reactions are inserted. This has the additional advantage that for the most part the same range of substances can be used for the detection of different molecules - only the first substance (often antibodies ) that forms a specific bond with the target has to be exchanged.
literature
- LM Prescott, JP Harley, DA Klein: Microbiology. 5th edition. McGraw Hill, Boston 2002, ISBN 0-07-232041-9 .