Biotinylation specifically refers to the process of attaching biotin to proteins or other macromolecules, including nucleic acid, functional groups and residues, in which antibodies are one of the most widely used targets.

Biotin, also known as vitamin H or vitamin B₇, is a water-soluble vitamin found naturally in eggs, dairy products, salmon, liver, etc. It influences on several biological processes within the human body, such as cell growth, fatty acids production, and fats and amino acids metabolism.

Due to attractive characteristics like small size, stability, specificity, and interaction with avidin or streptavidin, researchers extensively use biotin to label proteins, for example, antibody biotinylation, aiming at purification, detection, and cell sorting.

Mainly two approaches are applied to create biotinylated antibodies, one of which is the avidin-biotin complex (ABC), and the other is the labeled streptavidin-biotin (LSAB).

Avidin-biotin complex

The ABC method is broadly used for staining due to its modular and versatile with high sensitivity and low background. Most of the time, the ABC method is the first choice unless the avidin-biotin-enzyme complex is too large to pass through the tissue. There are some nonnegligible shortcomings also, such as nonspecific labeling caused by endogenous biotin and lower tissue penetration caused by large ABC complexes.

Labeled streptavidin-biotin

The LSAB method, on the contrary, generates smaller complexes that allow better tissue penetration and higher sensitivity. Alternatives to avidin can be used in this method to reduce background noise and further sensitivity.

Based on the strongest known non-covalent interaction between biotin and avidin (or streptavidin), the process of biotinylation is completed fast without affecting the original function of the antibody. And the protein-ligand interaction of biotinylated antibodies is unaltered by extremes of pH, high salt concentrations, denaturant agents, temperature, and organic solvents.

In general, proteins can be biotinylated chemically or enzymatically. Compared with chemical methods, Avi-tag biotinylation holds obvious advantages. As random biotinylation of the protein surface, proteins could become inactivated in the process of chemical biotin-labeling, while the single and unique Avi-tag site enables the protein to be easily and efficiently biotinylated in vivo or in vitro. What's more, the Avi-tag biotinylation reaction is more gentle with highly specific labeling.

Biotinylated antibodies are designed to detect low-abundance proteins. Biotinylation also contributes to non-radiative labeling methods of proteins and protein purification, with increasing applications in medical and industrial fields, including biosensors, diagnosis, proximity assay, and drug screening.

A variety of biotinylated reagents allow different functional group specificities and different solubility characteristics, which substantially expands the range of applications for avidin-biotin chemistry research.

The wide adoption of biotin brings about many companies that have long engaged in model organism antibody production, engineering, and manufacturing. Creative Biolabs is one of the most prominent contract research organizations (CROs), presenting a wide range of biotinylated proteins along with Avi-tag biotinylated antibody services for researchers.