Biotinylation of the Oxidized Antibody

1. Prepare the labeling reagent stock solution. Biotin, biocytin, and desthiobiotin hydrazide derivatives are typically used at a labeling reagent: antibody MR of 20-25. Biotin and desthiobiotin hydrazide stock solutions should be prepared at 10-20 mg/mL in DMSO or DMF while biocytin hydrazide can be dissolved in the reaction buffer. Use Eqs. 1-3 in Subheading 3.1.1. to calculate the amount of labeling reagent needed. (See Note 6 for information about reusing labeling reagent stock solutions.)

2. Add the labeling reagent to the oxidized antibody. The appropriate volume of hydrazide reagent should be added drop-wise to the oxidized antibody while stirring.

3. Incubate the stirring reaction mixture for 120 min at room temperature.

4. Desalt the biotinylated antibody by dialysis or gel filtration. Note that after hydrazide-based biotinylations, many researchers feel that it is necessary to stabilize the linkages between the hydrazides and aldehydes by treating the conjugate with sodium cyanoborohydride.

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