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In this final phase, we will demonstrate signal development using the most common,
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most sensitive and most inexpensive detection method the
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electrochemiluminescence (or ECL) reaction.
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This method utilizes the HRP enzyme,
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which was conjugated to the secondary
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to catalyze the ECL reaction and produce light.
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A light is then gathered onto x-ray film and developed or digitized with the
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aid of a specialized camera sensitive enough for this application.
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We start by mixing equal parts ECL reagents in a one-to-one ratio
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according to the manufacturer's instructions.
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We will incubate the membrane for 3-5 minutes without agitation.
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After incubation, decant ECL mixture and use a laboratory wipe to wipe off excess
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solution
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from the corner of the membrane.
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Place the membrane in a clear plastic wrap such as a sheet protector to
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prevent drying.
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Avoid letting the membrane completely dry out.
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We can now use a roller to push out any bubbles or any excess solution.
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Immediately develop the membrane.
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Both film and camera systems allow you to manually adjust the exposure time
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in order to ensure a picture perfect Western Blot. Relative band densities can
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now be quantified with commercially available software.
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Proper molecular weight can also be verified by comparing band sizes to the
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molecular weight ladder.
