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Chemiluminescent detection is the method of choice for Western blot analysis because of its signal output, high sensitivity and versatility for use with film or digital imagers. This approach requires a membrane probed with an antibody that is conjugated to an enzyme, typically horseradish peroxidase or HRP, and a chemiluminescent substrate. These substrates are usually 2-component systems consisting of a stable peroxide solution and a luminol or acridan solution to provide enhanced chemiluminecence, or ECL. While commercial ECL substrate kits vary in sensitivity,they all follow a general protocol. First, combine the luminol or acridan solution and the peroxide at the ratio indicated by the manufacturer. Next, pour the solution onto a membrane probed with an HRP-conjugated antibody. The luminol or acridan is oxidized by the peroxide in the presence of the HRP enzyme, resulting in the emission of chemiluminescent light. After incubating the membrane with the substrate for 3-5 minutes, r
