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Cyanagen has a product line of amine-reactive BIOTINYLATION REAGENTS for modifying biomolecules such as proteins and amine-modified nucleic acids for Biotin/Streptavidin applications. These reagents are divided into two groups based on water solubility: NHS and sulfo-NHS.
|B101N||Sulfo NHS-Biotin||Water soluble||443.43||16.1|
|B102N||Sulfo NHS-LC-Biotin||Water soluble||556.60||22.4|
D-biotin is a small molecule (MW 244.3) present in minute amounts in every living cell as a growth factor (vitamin H or Coenzyme R). It plays an indispensable role in numerous carboxylation reactions. Biotin combines with a protein, Avidin (MW 60.000), present in raw egg white and becomes inactive. The dissociation constant of the egg white Avidin/Biotin complex is among the lowest known (Kd = 10-15 M).
A perfect fit occurs between the bicyclic ring of Biotin and a pocket of the tetrameric protein.
The valeric acid side chain of Biotin is not directly involved in the bond. Thus, it can be activated or modified without altering or changing the binding affinity toward Avidin. An analogous interaction takes place between Biotin and the protein Streptavidin (MW 60.000, isolated from Streptomyces Avidinii).
Proteins or other molecules can be labeled with Biotin in a wide variety of methods. This process is called biotinylation. Since Biotin is a relatively small molecule, large macromolecules are hardly affected by the labeling. Biotinylation reagents are designed to form covalent bonds with common functional groups, in particular amino (-NH2), carboxylic (-COOH), aldehydes (-CHO) and thiols (–SH). The amine group is the most common target for modifying protein molecules because it is abundant in the majority of proteins due to the presence of lysine. Amine-reactive biotinylation reagents can be divided into two groups based on water solubility: NHS-esters and sulfo-NHS-esters.
NHS-esters of Biotin are water-insoluble and membrane-permeable because they do not possess a charged group. They may be used for biotinylating internal as well as external cellular components.
Sulfo-NHS-esters are water soluble and useful for applications that cannot tolerate organic solvents. Sulfo-NHS-esters of Biotin are recommended for use as cell surface biotinylation reagents.
Since the binding sites for Biotin are buried deep inside Avidin and Streptavidin (about 9 Ǻ below the protein surface) spacers improve both the accessibility and reaction rate of biotinylated compounds with respect to Avidin and Streptavidin, often enhancing the sensitivity of assay systems.
The most common spacer group is aminocaproic acid (LC), which increases the length of the side arm by about 9 Å.