Product Description of Amine-Reactive Biotins
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
-OSu or Biotin-(AC5
-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
-OSu is utilized as the biotin labeling agent in the Biotin Labeling Kit-NH2
Labeling Procedure for Protein
1. Prepare 50 mmol/l of biotin labeling reagent using DMSO (ex. 5.7 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.
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.
1) P. Kongtawelert and P. Ghosh, A New Sandwich-ELISA Method for the Determination of Keratan Sulphate Peptides in Biological Fluids Employing a Monoclonal Antibody and Labelled Avidin Biotin Technique., Clin. Chem. Acta,, 1990, 195, 17 .
2) G. Paganelli, S. Pervez, A. G. Siccardi, G. Rowlinson, G. Deleide, F. Chiolerio, M. Malcovati, G. A. Scassllati and A. A. Epenetos, Intraperitoneal Radio-localization of Tumors Pre-targeted by Biotinylated Monoclonal Antibodies, Int. J. Cancer, 1990, 45, 1184.
3) C. Wagener, U. Kruger and J. E. Shively, Selective Precipitation of Biotin-labeled Antigens or Monoclonal Antibodies by Avidin for Determining Epitope Specificities and Affinities in Solution-phase Assays, Methods Enzymol., 1990, 184, 518 .
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.