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Chemiluminescent HRP Detection for Western Blotting

Protocol Background

This protocol is presented as a translation from a sheet of paper I received from a colleague. It describes a luminol-based chemiluminescent protocol for detecting horseradish peroxidase-conjugated antibodies during Western immunodetection. This protocol serves to replace the commercially available "ECL" and "ECL PLus" technologies sold and licensed by GE Healthcare. The Protocol I obtained is titled "ECL SOUTHERN / WESTERNS DETECTION REAGENTS" with the footnote "adapted from St. Geme and Miller lab protocols 1/4/01 by her highness Ms. J. Sexton". I don't know who these folks are, but thank you very much, you save us a lot of money.

Recipes of Stocks

500 mM Tris-Cl, pH 8.5 , prepare 500 mLs or so, it will last a long time.

If you need help with this, you shouldn't be doing Westerns. Diluting Tris stocks substantially will change the pH. Also, be mindful of the temperature when you pH a Tris solution.

250 mM Luminol

Buy Luminol (C8H7N3O2), and dissolve it in DMSO. You just need 5-10 mLs.

CAS # 521-31-3 AKA: 3-Aminophthalhydrazide AND 5-Amino-2,3-dihydro-1,4-phthalazinedione

90 mM ρ-coumaric acid, 5-10 mLs.

CAS # 501-98-4

Also dissolved in DMSO.

30-35 % hydrogen peroxide, remove an aliquot from the stock bottle and put it in a microfuge tube to be kept wight he other reagents.

There are numerous sources available. Some solutions that are 33% are cheaper than 30%, so you can buy those and adjust the volumes accordingly. Get clean stuff and keep your fingers out of it. If this reagent goes bad, the system won't work. It is a good idea to have aliquots replaced regularly.

Keep these in the dark when not in use, we keep our in a dark cabinet at room temperature for months. If the kit stops working, usually the peroxide aliquot is bad. Precipitates may form in the luminal tube, it still works okay.

Luminescence Solution

This was updated to make the mixing more straightforward.

Pour ~2 mL of 500 mM Tris solution into a 15 mL conical tube.

Squirt clean water into the tube to make 10 mLs at ~100 mM Tris.

Add 50 μL of the Luminol stock (1.25 mM final, it will cloud briefly before you mix it into solution)

Add 25 μL of Coumaric acid stock and mix (0.225 mM final, this can sit on the bench while you finish washing your membrane)

Just before you pour off the last wash, add 3 μL of hydrogen peroxide and mix well, the reagent is now active.

If you are unsure about the reagents, you can mix a sacrificial tube and pipette in ~0.1 μL of an HRP-conjugated antibody. The tube should glow in the dark for a few minutes.


You will have more consistent results if you have the solutions at room temperature before using, but it is not essential.

During the last wash of your membrane, mix the "Luminescence Solution" described above. For mini-gels, making ~10 mLs is plenty, you can adjust the volumes to suit your membrane sizes.

Drain the last wash off, do not let the membrane dry.

Pipette/pour the active solution onto the blot.

Mix it a few times by tilting the blot and pipetting the solution over the surface. Do NOT touch the membrane with your tip.

After a few minutes (~1-5 min, make sure you don't see areas drying), lift the membrane out of the solution either with tweezers or by sliding a pipette tip under the edge and grabbing a corner with gloves, briefly let the gross excess drip off, and place it face-down on clear plastic wrap. (If you have a luminescence camera, you can just place the un-wrapped membrane on a solid support face up and image the Western).

Fold the wrap to prevent the excess solution from leaking and expose to film in a dark room.

Expose according to experience. What this means is the signal intensity can vary greatly depending on (1) how much target protein you loaded on the gel, (2) How well presented the epitopes are on the membrane after transfer, (3) how specific/strong your antibody is, (4) how well conjugated your antibody is, (5) how old the reagents are... you get the idea. I start with a 15 second exposure, then quickly move the blot to an unexposed area for a minute, then develop the film. Sometimes even that is too long of an exposure, cut back on something to weaken the signal the next time you do one. Be aware that the signal intensity is changing for each band as the reagent sits on the HRP. Where there is a lot of enzyme, the center of the bands will burn out.

Our camera imaging system automatically collects longer and longer exposures and we have abandoned film.


The protocol was edited to condense the procedure in the original protocol.