The easiest and most commonly used method for measuring the average DOL is based on displacement of the deep-orange dye HABA (4'-hydroxyazobenzene-2-carboxylic acid) by biotin from avidin or streptavidin (12). When HABA binds to these proteins, a dye absorbance peak at 500 nm (A500) appears. As increasing amounts of biotinylated protein are added to the complex, the biotin displaces the weaker-binding HABA from the protein. This results in a decrease in A500, which is proportional to the amount of biotin added. In one variation of the assay, the biotinylated antibody is first digested to completion with the protease pronase to generate many small protein fragments, some of which are biotinylated. This procedure permits one to determine the average total number of biotins conjugated to the antibody. However, using the intact, biotinylated antibody in the HABA assay gives an estimate of the average number of accessible biotins, which is usually lower. The latter more realistically reflects how readily the labeled antibody can be recognized by reporter group-labeled biotin-binding proteins.
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