In biochemistry, biotinylation is the process of covalently attaching biotin to a protein, nucleic acid or other molecule. Biotinylation is rapid, specific and is unlikely to disturb the natural function of the molecule due to the small size of biotin (MW = 244.31 g/mol). Biotin binds to streptavidin and avidin with an extremely high affinity, fast on-rate, and high specificity, and these interactions are exploited in many areas of biotechnology to isolate biotinylated molecules of interest. Biotin-binding to streptavidin and avidin is resistant to extremes of heat, pH and proteolysis, making capture of biotinylated molecules possible in a wide variety of environments. Also, multiple biotin molecules can be conjugated to a protein of interest, which allows binding of multiple streptavidin, avidin or neutravidin protein molecules and increases the sensitivity of detection of the protein of interest. There is a large number of biotinylation reagents available that exploit the wide range of possible labelling methods. Due to the strong affinity between biotin and streptavidin, the purification of biotinylated proteins has been a widely used approach to identify protein-protein interactions and post-translational events such as ubiquitylation in molecular biology.

Proteins can be biotinylated chemically or enzymatically. Chemical biotinylation utilises various conjugation chemistries to yield nonspecific biotinylation of amines, carboxylates, sulfhydryls and carbohydrates (e.g., NHS-coupling gives biotinylation of any primary amines in the protein). Enzymatic biotinylation results in biotinylation of a specific lysine within a certain sequence by a bacterial biotin ligase. Most chemical biotinylation reagents consist of a reactive group attached via a linker to the valeric acid side chain of biotin. As the biotin binding pocket in avidin / streptavidin is buried beneath the protein surface, biotinylation reagents possessing a longer linker are desirable, as they enable the biotin molecule, once it has been attached to its target, to be more accessible to binding avidin/streptavidin/Neutravidin protein. This linker can also mediate the solubility of biotinylation reagents; linkers that incorporate poly(ethylene) glycol (PEG) can make water-insoluble reagents soluble or increase the solubility of biotinylation reagents that are already soluble to some extent.

In contrast to chemical biotinylation methods, enzymatic biotinylation allows biotin to be linked at exactly one residue present in the protein. This biotinylation reaction can also go to completion, meaning that the product is generated with high uniformity and can be linked to streptavidin in a defined orientation e.g. for MHC multimers. Enzymatic biotinylation is most often carried out by E. coli biotin holoenzyme synthetase, also known as biotin ligase (BirA, P06709).

The most common way of targeting a protein of interest is by fusing the protein at its N-terminus, C-terminus or at an internal loop to a 15 amino acid peptide (GLNDIFEAQKIEWHE), termed AviTag or Acceptor Peptide (AP). Once tagged, the protein is then incubated with BirA allowing biotinylation to take place in the presence of biotin and ATP. Enzymatic biotinylation can be carried out in vitro but BirA also reacts specifically with its target peptide inside mammalian and bacterial cells and at the cell surface, while other cellular proteins are not modified. Enzymatic biotinylation can also take place in vivo typically through the co-expression of an Avitag tagged protein and BirA.

The natural substrate of BirA is the biotin carboxyl carrier protein (BCCP). Before smaller tags were discovered, a protein needed to be fused to the entire BCCP to be targeted. A protein fused by BCCP can be recognized by biotin molecules in vivo and attach to it. A few other small tags have been used before AviTag, but AviTag is the most efficient so far.

The most common targets for modifying protein molecules are primary amine groups that are present as lysine side chain epsilon-amines and N-terminal α-amines. Amine-reactive biotinylation reagents can be divided into two groups based on water solubility.

N-hydroxysuccinimide (NHS) esters have poor solubility in aqueous solutions. For reactions in aqueous solution, they must first be dissolved in an organic solvent, then diluted into the aqueous reaction mixture. The most commonly used organic solvents for this purpose are dimethyl sulfoxide (DMSO) and dimethyl formamide (DMF), which are compatible with most proteins at low concentrations. Because of the hydrophobicity of NHS-esters, NHS biotinylation reagents can also diffuse through the cell membrane, meaning that they will biotinylate both internal and external components of a cell.

Sulfo-NHS esters are more soluble in water and should be dissolved in water just before use because they hydrolyze easily. The water solubility of sulfo-NHS-esters stems from their sulfonate group on the N-hydroxysuccinimide ring and eliminates the need to dissolve the reagent in an organic solvent. Sulfo-NHS-esters of biotin also can be used as cell surface biotinylation reagents, because they do not penetrate the cell membrane.