Product Description of Amine-Reactive Biotins
The avidin-biotin system has many applications in immunology and histochemistry. The interaction between avidin and biotin is remarkably strong with a dissociation constant on the order of 10-¹⁵ M. Biotin is usually added to primary or secondary antibodies such as anti-IgG and anti-IgM. After preparing the antigen-antibody complex with the biotin-labeled antibody, colorimetric, or fluorometric detection of the antigen is performed using enzyme or fluorescein-labeled avidin or streptavidin. Succinimidyl ester biotins react with primary and secondary amines, such as amino acids and proteins, at pH 7-9. Succinimidyl ester reacts with free amine groups to create a stable amide bond. Succinimidyl biotin reagents must be dissolved in DMSO, DMF, or alcohol. Stock solutions prepared with DMSO are stable for several months at -20ºC. Sulfo succinimidyl biotin reagents are soluble in water, so there is no need to use organic solvents such as DMF or DMSO. IgG prepared using biotin with a longer spacer such as Biotin-(AC₅)₂-OSu or Biotin-(AC₅)₂-Sulfo-OSu, has a better signal-to-noise ratio. The longer spacer enables streptavidin or anti-biotin IgG to recognize biotin without structural inhibition. Therefore, Biotin-(AC₅)₂-OSu is utilized as the biotin labeling agent in the Biotin Labeling Kit-NH₂.

Reaction Scheme

Labeling Procedure for Protein

1. Prepare 50 mmol/l of biotin labeling reagent using DMSO (ex. 4.5 mg/200 ul for 50 mmol solution) *Once biotin labeling reagent is solved in water, please mix it to sample protein immediately.
2. Prepare protein solution using Bicine buffer(pH8.5, 10 mmol/l).
3. Add the prepared solution of biotin labeling reagent to protein solution and mix them. Please mix protein and biotin as the molar ratio of 1:2 - 1:10 (protein : biotin).
4. Incubate the mixed solution at 25ºC for 2-4 hour.
5. Remove excess biotin labeling reagent using gel filtration or dialysis.

<Caution>
When it is difficult to take out all the powder from the container,
please add the solvent into a container and dissolve it before its use.

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4) D. M. Boorsma, J. Van Bommel and E. M. Vander Raaij-Helmer, Simultaneous Immunoenzyme Double Labelling Using Two Different Enzymes Linked Directly to Monoclonal Antibodies or with Biotin-avidin, J. Microscopy, 1986, 143, 197.
5) A. Komura, T. Tokuhisa, T. Nakagawa, A. Sasase, M. chihashi, S. Ferrone and Y.Mishima, Specific Killing of Human Melanoma Cells with an Efficient 10B-compound on Monoclonal Antibodies, Pigment Cell Res., 1989, 2, 259.
6) R. Rappuoli, P. Leoncini, P. Tarli and P. Neri, Competitive Enzyme Immunoassay for Human Chorionic Somatomammotropin Using the Avidin-biotin System, Anal. Biochem., 1981, 118, 168.