Biotinylation is the process of labeling biotin to a protein, nucleic acid, or other molecules. Biotinylated protein can be a useful tool for a variety of assay formats. There are two key benefits of labeling protein/antibody with biotin.
Biotin has a small molecular weight of 244.3 Da and therefore is easy to label target protein without impacting its structure and bioactivity.
Biotin binds to avidin/streptavidin with an extremely high affinity, high specificity and fast speed. These characteristics are ideal for assay development.
Biotinylation is frequently used to identify protein-protein interaction, post-translational events, or drug targets. The common applications of biotinylated protein include enzyme-linked immunosorbent assay (ELISA), western blots, flow cytometry, and surface plasmon resonance (SPR).
Example of using biotinylated protein in ELISA, to examine the protein binding activity
$2,500 All-Inclusive Biotinylation Service
From gene synthesis, protein expression, to biotinylation.
Provide 3 types of protein biotinylation strategy: in-vitro chemical biotinylation, in-vitro or in-vivo enzymatic biotinylation.
With 100% success rate of biotinylation, we guarantee to label the custom protein with higher than 0.5 biotin ratio.
In chemical biotinylation, N-hydroxysuccinimide (NHS) ester is added to the target protein, enabling the biotinylation of primary amine group within the lysine side chain and the N-terminus of protein. As a result, the labeling is non-specific and occurs on multiple sites. Chemical biotinylation can increase the detection sensitivity of biotin; however, the attached biotin level varies within the protein sample.
Enzymatic Biotinylation (Site-specific label)
Enzymatic Biotinylation specifically attaches biotin to the lysine within AviTag on each protein. Therefore, the labeled proteins are homogeneous and can bind to streptavidin in a defined orientation. To perform enzymatic biotinylation, our protein team will design and add a 15 amino acid AviTag to the target protein during the construct. The enzymatic biotinylation then is carried out by E. coli biotin ligase BirA, which can be either added in vitro or co-expressed in E. coli with the target protein.
Determine the biotinylation level
At Bon Opus Biosciences, we use HABA assay to estimate the biotinylation level. HABA dye binds to avidin/streptavidin and generates a characteristic absorbance of 500 nm. When biotinylated protein is introduced, the biotin can displace the dye, causing a decrease in absorbance can be detected. Therefore, the biotinylation level can be determined based on the change of absorbance.