ReadiView™ Biotinylation Technology

Biotin is a vitamin (Vitamin H, Vitamin B7, Coenzyme R) that is present in small amounts in all living cells and is critical for a number of biological processes including cell growth and the citric acid cycle. Biotin is abundant in certain plant and animal tissues such as corn kernels, egg yolk, brain, liver and blood. The valeric acid side chain of the biotin molecule can be derivatized in order to incorporate various reactive groups that facilitate the addition of a biotin tag to other molecules. Because biotin is relatively small (244.3 Daltons), it can be conjugated to many proteins and other molecules without significantly altering their biological activity. The highly specific interaction of biotin-binding proteins with biotin make it a useful tool in assay systems designed to detect and target biological analytes.

Once biotin is attached to a molecule, the biotin tag can be used to facilitate affinity purification of that molecule using an immobilized biotin-binding protein. Alternatively, a biotinylated molecule can be immobilized through interaction with a biotin-binding protein, and then used to affinity purify other molecules that specifically interact with it (i.e., co-immunoprecipitation or pull-down assays). In the context of immunohistochemistry and immunoblotting, biotin is most often conjugated to primary or secondary antibodies, and the biotin tag is then detected with a biotin-binding protein that is conjugated to an enzyme, fluorophore or other reporter molecule. Many proteins, such as antibodies, can be labeled with several biotin tags, each able to be bound by a biotin-binding protein. An optimized biotin-to-probe ratio can greatly increase the signal output of a detection system making it possible to create very sensitive assays. Although a large number of biotin-labeled bioconjugates are commercially available, the accurate determination of biotinylation degree (ratio of biotin/biopolymer) is still a great challenge for biochemists. HABA is still predominantly used for determining the degree of biotinylation (through its absorption with the extinction coefficient = 34,000/M^-1cm^-1). When a biotin-containing sample is added, the biotin binds strongly to avidin and displaces the weakly bound HABA. The resulting decrease in absorbance relates to the amount of biotin. However, there are many factors that affect the accuracy of the HABA method, making it unreliable for many biotin-labeled conjugates.

ReadiView™ biotin contains a specially designed color tag (CT) that makes the biotinylation degree readily accessible by simply calculating the corrected absorption ratio of 280 nm/385 nm. The specially designed tag has very minimal effect on the biotin binding affinity, and its absorption maximum is designed to make the tag have minimal quenching effect on most fluorophores that are used for labeling avidins.

ReadiLink™ Protein Biotinylation Kit (Cat# 5521) is primarily used to prepare of biotin-labeled IgG for enzyme immunoassays (EIA). The kit uses our ReadiView™ Biotin Succinimidyl Ester (Cat# 3059) that reacts with an amino group of IgG and other biomolecules. Our unique biotin contained in the kit carries a color tag for indicating the degree of biotinylation, thus eliminating the need for the troublesome HABA biotinylation determination. The kit contains all of the necessary reagents for labeling and purification. Based on our in-house experiments, 5 to 8 biotin molecules can be conjugated to each IgG molecule using ReadiLink™ Protein Biotinylation Kit.