Several varieties of Thermo Scientific Pierce ECL and SuperSignal chemiluminescent HRP substrates are available that provide different levels of sensitivity for chemiluminescence western blotting. Because its reaction rate remains linear, detection sensitivity can be improved by simply allowing a reaction to proceed for a longer time period.Īs HRP is the most popular enzyme used in western blotting it will be discussed throughout this article as our example. As a label for Western blotting, AP offers a distinct advantage over other enzymes. AP has optimal enzymatic activity at a basic pH (pH 8–10) and can be inhibited by cyanides, arsenate, inorganic phosphate and divalent cation chelators, such as EDTA. Alkaline phosphatase (AP) catalyzes the hydrolysis of phosphate groups from a substrate molecule resulting in a colored or fluorescent product and the release of light as a byproduct of the reaction. Because of the relatively small size of the HRP enzyme, further increases in sensitivity may be achieved by using poly-HRP conjugated secondary antibodies and may eliminate the need for using ABC-type amplification systems for some researchers. In addition, its high turnover rate, good stability, low cost, and wide availability of substrates makes HRP the enzyme of choice for most applications. Antibody-HRP conjugates are superior to antibody-AP conjugates with respect to the specific activities of both the enzyme and antibody. HRP functions optimally at a near-neutral pH and can be inhibited by cyanides, sulfides and azides. Horseradish peroxidase (HRP) catalyzes the oxidation of substrates by hydrogen peroxide, resulting in a colored or fluorescent product and the release of light as a by-product of the reaction.
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