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 the 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 fluorophore-labeled avidin or streptavidin. Maleimide biotins react with thiol compounds, such as proteins or peptides with sulfhydryl groups, at pH 7-7.5. Maleimide reacts with sulfhydryl group to create a thioether bond. Though other maleimide biotin reagents must be dissolved in DMSO, DMF, or alcohol, Biotin-PE-maleimide can be solubilized in PBS at pH 7.4 to prepare 2 mM solution without using an organic solvent. The reactivity of maleimide with sulfhydryl groups is higher than that of bromoacetamide, so the required concentration of maleimide biotin is much lower than that of bromoacetamide biotins. Stock solutions of Biotin-PE-maleimide and Biotin-PEAC5-maleimide in DMSO are stable for one year at -20oC.
Labeling Procedure for Reduced IgG
1. Prepare 10 mM of the biotin labeling reagent using DMSO.
2. Prepare 100 μl of 1 mg per ml reduced IgG/ml buffer solution which does not contain any large molecules with SH groups. Reduced IgG can be prepared by TCEP (tricarboxyethylphosphine), DTT or 2-mercaptoethylamine.
3. Add 1-5 μl of biotin labeling reagent DMSO solution to the IgG buffer solution and incubate at 37 oC for 1 hour.
4. Remove excess biotin labeling reagent using a gel column or a Filtration tube.
5. Prepare solutions for further experiment using an appropriate buffer such as PBST (0.05% Tween 20/PBS).
E. Muneyuki, et al., Biophys. J., 92, 1806 (2007).