1. Biotinylate the reduced antibody. Thiol-reactive biotinylation reagents are usually used at labeling reagent: antibody MR of 16-20. Use Eqs. 1-3 in Subheading 3.1.1. to determine the appropriate amount of labeling reagent to add to the reduced antibody. Slowly add the labeling reagent solution drop-wise to the reduced antibody while stirring. (See Note 6 for information about reusing biotinylation reagent stock solutions.)
2. Incubate the reaction mixture for 90 min at room temperature. If the labeling reagent is iodoacetyl-LC-biotin (Pierce), both the reagent and the reaction mixture should be protected from light.
3. Remove unreacted labeling reagent from the biotinylated antibody by dialysis, gel filtration, or with a spin column.
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