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 manufacturer's 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 membrane.
Both film and camera systems allow you to manually adjust the exposure time
in order to ensure a picture perfect Western Blot. Relative band densities can
now be quantified with commercially available software.
Proper molecular weight can also be verified by comparing band sizes to the
molecular weight ladder.