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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 replaces 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

250 mM Luminol

Buy Luminol (C8H7N3O2), and dissolve it in DMSO. CAS # 521-31-3 AKA: 3-Aminophthalhydrazide AND 5-Amino-2,3-dihydro-1,4-phthalazinedione Keep it in the dark when not in use (refrigerator is fine if your Fridge Penguin turns out the light when you close the door).

1M Tris-Cl, pH 8.5

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.

90 mM ρ-coumaric acid

CAS # 501-98-4

Also dissolved in DMSO. Keep this dark as well.

30% hydrogen peroxide

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 fresh aliquots used avery few months.

Recipes of Working Solutions

Detection Solution 1

2.5 mM Luminol

400 μM ρ-coumaric acid

100 mM Tris-Cl, pH 8.5

all from above stocks.

You can make a hundred mLs, but making 40 or 50 mLs in a 50 mL Falcon tube is easier.

Detection Solution 2

100 mM Tris-Cl, pH 8.5

Just dilute the stock you made, this one baffles me, see comments below.

Active Detection Solution

5 mL of Detection Solution 1

5 mL of Detection Solution 2

3.1 μL of hydrogen peroxide solution (use less if using a more concentrated stock)