Moore:Chemiluminescent: Difference between revisions
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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). | 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). | ||
<u>1M Tris, pH 8.5 </u> | <u>1M Tris-Cl, pH 8.5 </u> | ||
If you need help with this, you shouldn't be doing Westerns | 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. | ||
<u>90 mM ρ-coumaric acid</u> | <u>90 mM ρ-coumaric acid</u> |
Revision as of 09:03, 12 November 2008
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)