The procedures for coupling of biotin succinimide ester to proteins are described in Section 10.3. When biotinylating antibodies, the most important parameter is the ratio of biotin to IgG. If too little biotin is used, the sensitivity will be poor. If too much is used, there comes a point where the antibody may become inactivated and/or aggregated, with reduction in both sensitivity and specificity (Wadsley and Watt, 1987; Muzykantov et al., 1995). Because of the competing hydrolysis reaction, the amount of biotin succinimide ester to be added to the antibody must be matched to the antibody concentration as well as its mass. Unlike fluorochromes, biotin does not absorb light in the visible region of the spectrum, and there is no easy way to measure the biotin:protein ratio of conjugated antibodies. It is therefore best to adhere closely to the conditions described in the following section.
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