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.
Labeling Procedure for IgG
1. Prepare 10 mM of the biotin labeling reagent using DMSO.
2. Prepare 100 μl of 1 mg per ml IgG buffer solution (pH 7.5-8.5) that does not contain any large molecules with amine compounds.
3. Add 1-5ml biotin labeling reagent DMSO solution to the IgG buffer solution and incubate at 37ºC for 1 hour.
4. Remove excess biotin labeling reagent using gel filtration or dialysis.
5. Prepare solutions for further experiment using an appropriate buffer such as PBST (0.05% Tween 20/PBS).
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