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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, pH 8.5

If you need help with this, you shouldn't be doing Westerns

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