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.