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In this final phase, we will demonstrate signal development using the most common, most sensitive and most inexpensive detection method the electrochemiluminescence (or ECL) reaction. This method utilizes the HRP enzyme, which was conjugated to the secondary to catalyze the ECL reaction and produce light. A light is then gathered onto x-ray film and developed or digitized with the aid of a specialized camera sensitive enough for this application. We start by mixing equal parts ECL reagents in a one-to-one ratio according to the manufacturers instructions. We will incubate the membrane for 3-5 minutes without agitation. After incubation, decant ECL mixture and use a laboratory wipe to wipe off excess solution from the corner of the membrane. Place the membrane in a clear plastic wrap such as a sheet protector to prevent drying. Avoid letting the membrane completely dry out. We can now use a roller to push out any bubbles or any excess solution. Immediately develop the