Addition of Reactive Biotin

1. Stir the antibody solution gently at room temperature prior to adding the labeling reagent. If the antibody is already in a buffer at pH 8.08.3, slowly add the volume of labeling reagent solution determined using Eq. 3 drop-wise while stirring. If the buffer pH is not in this range, it should first be adjusted as discussed in Note 3 before adding the labeling reagent. (See Note 6 for information about reusing labeling reagent stock solutions.)

2. Incubate the reaction mixture with stirring for 60-90 min at room temperature.

3. Remove the unconjugated biotinylation reagent by dialysis or gel filtration (see Note 4).

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