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1000
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Abstract/Summary
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The extraordinarily strong interaction between streptavidin (SA) and biotin (the dissociation constant Kd ∼ 10−14 M) has been exploited extensively in biotechnologies [1]. One such application is using horseradish peroxidase (HRP)-conjugated SA to detect biotinylated proteins in western blot (WB). For routine WB using an antibody to detect the target protein, 5% (w/v) low-fat dry milk dissolved in tris-buffered saline/Tween 20 (TBS/T) solution is the most common blocking and antibody incubation buffer. However, milk contains biotin, which will competitively inhibit the interaction between SA and biotinylated proteins [2]. For WB using SA-HRP, common practice is to use either bovine serum albumin (BSA) to substitute milk, or reduce milk to 1%; less commonly, a specialized commercial blocking agent absent of biotin is used. However, each of these methods has a drawback: replacing milk with BSA not only increases the cost but also results in heavy background, while using 1% milk results in insufficient blocking and loss of specific signals (see Figure 1); the loss of signals could be overcome by increasing the concentration of SA-HRP, but this will increase the cost and potentially further increase the background; a specialized blocking reagent is generally not readily available for many laboratories and will increase the cost markedly compared with milk.
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