What is Biotinylation?

What is Biotinylation?

Published by BroadPharm on March 12, 2020

What is Biotinlyation?

Biotinylation, also known as biotin labeling, is the process of covalently attaching biotin(s) to biomolecules: such as proteins, antibodies, peptide, oligonucleotide, and other macromolecules. The reaction is rapid, specific and is unlikely to disturb the natural function of the biomolecules due to the small size of biotin (MW = 244.31).

Biotin specifically binds to streptavidin and avidin to form a complex with an extremely high affinity (Kd, ~ 10-14 mol/L) and fast on-rate. The complexes are very stable under even extreme conditions such as high/low pH, high temperature, high salt concentrations, etc.

Biotin-avidin and streptavidin systems are widely used in detection and separation of target antigens/cells. These applications include immunoassays (ELISAs and Westerns being the most popular applications), affinity chromatography, pull-down assays, supramolecular construction, targeting of cancer cells for drug delivery, and many others. Recently, there are increasing application demands for protein/cell separation based on biotin-(strept)avidin interaction and magnetic beads.

biotin labeled antibody binds with streptavidin or avidin (four binding site available, only one is shown to binding to biotin).
Figure 1. biotin labeled antibody binds with streptavidin or avidin (four binding site available, only one is shown to binding to biotin).

Biotinylation Chemistry

For biotinylation chemistry, the most common reactions involve amines with biotin-NHS ester and click chemistry of azide with an alkyne (e.g. DBCO), as shown in Figure 2.

NHS-amine chemistry (A) and click chemistry between azide-DBCO
Figure 2. NHS-amine chemistry (A) and click chemistry between azide-DBCO (B).

Biotinylation Reagent in Biotin Labeling

The selection of biotinylation reagent should consider a few factors: target functional group, water solubility, cell membrane permeability, cleavability, and length of the reagent.

The functional groups can be click chemistry reactive, amine reactive, carbonyl reactive, carboxyl reactive, and sulfhydryl reactive (Figure 3). There are also reversible and cleavable biotinylation reagents to help with the specific elution of biotinylated proteins.

Pegylated biotin reagents are particularly attractive due to their water solubility, no toxicity, and low immunogenic properties. Monodispersed PEGs have a well-defined chain length, allowing for specific biotin-based complexes to be designed and studied.

Examples of BroadPharm's Pegylated biotinylation reagent with R 1 and R 2 options for functional groups
Figure 3. Examples of BroadPharm's Pegylated biotinylation reagent with R 1 and R 2 options for functional groups.

In addition, BroadPharm provides desthiobiotin products for special application, such as affinity purification. Desthiobiotin is a modified form of biotin that binds less tightly to avidin and streptavidin than biotin while still providing excellent specificity. Unlike biomolecules that are labeled with biotin, proteins and other targets labeled with desthiobiotin can be eluted under a soft, mild elute conditions to avoid denaturing the protein of interest.

BroadPharm is a leader of biotinylation reagents that help advance our customer's research, and offers a variety of pegylated and non-pegylated biotinylation reagents to meet your requirement.