Biotin is a water-soluble B vitamin, also called vitamin B7 and formerly known as vitamin H or coenzyme R, whose MW is 244.31 g/mol. Biotin is composed of a ureido ring fused with a tetrahydrothiophene ring. A valeric acid substituent is attached to one of the carbon atoms of the tetrahydrothiophene ring.
In biochemistry, biotinylation is the process of covalently attaching biotin to a protein, nucleic acid or other molecules. Because of the small size, biotin is unlikely to perturb the activity and natural function of the labeled molecule.
There are many methods for biotin labeling, such as enzymatic biotinylation and chemical biotinylation. Chemical biotinylation utilises various conjugation chemistries to yield nonspecific binding, including primary amine biotinylation, sulfhydryl biotinylation, carboxyl biotinylation, glycoprotein biotinylation, oligonucleotide biotinylation, and non-specific biotinylation.
After the biotinylation process, these biotin-labeled proteins demonstrate superb bioactivity and high detection sensitivity. These features make biotinylated proteins suitable for purification and detection, such as ELISA assays, ELISPOT assays, SPR, FACS analysis, dot blot, western blots and other immunoanalytical methods.
Avidin and Avi-biotinylated Protein
Avidin is a tetrameric biotin-binding protein, which contains four identical subunits, each of which can bind to biotin with a high degree of affinity and specificity. Biotin-binding to avidin is resistant to extremes of heat, pH and proteolysis, making capture of biotinylated molecules possible in a wide variety of environments. When constructing the expression vector, the Avi tag, a unique 15 amino acid peptide, is introduced into the recombinant protein. Then the single lysine residue in the Avi tag is enzymatically biotinylated by biotin ligase BirA. Because the biotinylation happened on the Avi tag, and therefore does NOT interfere the target protein’s natural binding activities. Avidin's affinity for biotin is exploited in wide-ranging biochemical assays, including western blot, ELISA, ELISPOT and pull-down assays. Besides, when immobilized on an avidin-coated surface, the protein orientation is uniform because the position of the Avi tag in the protein is precisely controlled.
Challenge and Advantage
However, there are still many challenges for generating high-quality biotinylated proteins, including insufficient labeling, high batch-to-batch variation, and unpredictable performance. To release you from laborious biotinylation process, Creative BioMart provides pre-biotinylated proteins to help save your time and money. We guaranty of these biotinylated proteins with the superb bioactivity and high sensitivity, closely natural conformation and modification, and high repeatability and stability.