biotinylated

Biotinylated describes a molecule that has had biotin covalently attached through chemical conjugation or enzymatic tagging. Biotin is a small, water-soluble vitamin that binds with exceptional affinity to streptavidin and avidin, enabling a widely used platform for detection, purification, and immobilization. The biotin–streptavidin interaction is extremely strong under most conditions, though reversible forms exist when competing ligands such as desthiobiotin are used.

Biotinylation can be achieved by several methods. Chemical biotinylation often employs NHS-biotin derivatives that react with primary amines on proteins (commonly lysine residues) or with accessible amino groups on other biomolecules. Sulfo-NHS variants are water-soluble and favored for aqueous reactions, while other reagents target thiols or carboxyl groups using suitable linkers. Enzymatic or site-specific approaches use biotin ligase (BirA) to attach biotin to a specific AviTag sequence, yielding homogeneous, well-defined labeling. The latter methods are particularly valuable when preserving the biological activity of the molecule is important.

Applications of biotinylated molecules are widespread. They include affinity purification using streptavidin or avidin matrices, pull-down assays, Western blot and ELISA detection, immunohistochemistry, and various forms of imaging using fluorescent streptavidin. Biotinylated nucleic acids, proteins, antibodies, and labeling reagents are common in research and diagnostics. In some cases, reversible labeling strategies (e.g., desthiobiotin) or site-specific, low-degree biotinylation are preferred to minimize functional disruption.

Considerations include control of labeling stoichiometry, avoidance of steric hindrance, and verification of preserved function. Selection of chemical versus enzymatic labeling, choice of linker length, and the potential impact on molecular interactions are important design factors in biotinylation experiments.