Biotin Azide Plus

Product Information

A biotin labeling reagent for biomolecules using copper catalyzed azide alkyne cycloaddition (CuAAC) with minimal copper concentrations

What is biotin

Biotin, also known as vitamin B7, is a sulfur and nitrogen containing bi-heterocyclus which is used as coenzyme in metabolism. Biotin is used as coenzyme for carboxylation enzymes such as Acetyl-CoA Carboxylase or Pyruvate Carboxylase. Next to its function as coenzym, biotin is known to undergo one of the strongest non-covalent bonds known today to the proteins avidin and streptavidin. The formation of this strong complex is utilized in many research fields in modern biochemistry such as in peptide/protein and nucleic acid research. Usually, streptavidin is used because it is better soluble at neutral pH and has less unspecific binding than avidin.

Applications of biotin

Peptide/protein research:

The biotin streptavidin complex is used for many applications in research. Biotinylation of peptides is widely used for affinity purification. Binding of the biotin marked proteins or complexes to magnetic beads or columns allows for a simple one-step purification. Additionally, it enables the detection of biotin labelled targets by conjugation with streptavidin conjugated reporters. As reporters mostly fluorophores or enzymes as horseradish peroxidase (HRP) are used. Biotinylation is often used in immunoassays such as ELISA, Western Blot or Immunohistochemistry. It also enables the enrichment of certain proteins as exemplary newly synthesized ones for mass spectroscopy analysis. Fixation of biotin tagged proteins on a Streptavidin coated surface allows performing binding assays to study protein interactions.

Nucleic acid research:

In nucleic acid research biotin labeling is mostly used for purification processes. Again, the biotin bonding to Streptavidin containing beads, columns or resins is utilized to link the nucleic acids to the solid support and separate them from impurities. Biotin containing probes can be used in in situ hybridization (ISH), Southern blots or Northern blots for binding to specific DNA/RNA structures. The probes can be visualized and localized afterwards by binding to Streptavidin based markers which again carry usually a dye or an enzyme for detection.

For all these techniques biotin labeled biomolecules are needed. A cheap and easy to perform technique to produce these, is click chemistry. For using click chemistry a modified biomolecule is needed carrying a terminal alkyne. Biomolecules carrying this modification are usually available for purchase. Biotin labelled Oligos/mRNA are directly available from baseclick as well as modified nucleic acids containing terminal alkynes for custom labeling. For other targets please contact us via support@baseclick.eu if you have any questions.

Chemical Properties of Biotin Azide Plus

Biotin Azide Plus is a chemical for biotin labeling under the mild conditions of copper catalyzed azide alkyne cycloadditon (CuAAC) click chemistry. The azide plus linker is specifically designed to enable click chemistry with reduced copper concentration. If the copper free strain promoted alternative should be used, the azide plus linker is not providing any advantage and e.g. Biotin-PEG4-Azide is a cost effective alternative.

Click chemistry is considered to be bioothogonal, meaning that the functional groups included in the reaction do not interfere with any of the functionalities of living organisms. Additionally, the azide plus moiety is able to chelate copper ions, increasing reaction speed by providing the catalyst while providing protection to copper ions for them to catalyze side reactions. This is possible due to the fact that the azide plus functionality is chelating copper. This allows also to reduce the copper concentration during the reaction while maintaining the same results without the need to mandatory use a copper chelating ligand as THPTA.

These chemical properties make Biotin Azide Plus the ideal choice for biotin labeling of all kinds of terminal alkyne modified sensitive biomolecules such as peptides/proteins and nucleic acids if the strain promoted variant should be circumvented. The strain promoted alkyne azide cycloaddition (SPAAC) has some disadvantages as producing two isomers or the need for bulky strained alkynes as DBCO groups.

Since Click Chemistry utilizes non-natural occurring modifications, the possible positions of biotin labeling can be strongly controlled unlike traditional biotin labeling processes as NHS ester chemistry.

LITERATURE

Essential requirement for JPT2 in NAADP-evoked Ca2+ signaling, G. S. Gunaratne et al., 2021, Science Signaling, Vol. 14(675), eabd5605.

https://doi.org/10.1126/scisignal.abd5605

TMEM132A, a novel Wnt signaling pathway regulator through Wntless (WLS) interaction, B. Li et al., 2020, Frontiers in Cell and Developmental Biology, Vol. 8, 599890.

https://doi.org/10.3389/fcell.2020.599890