How To Make Your Own ECL
ECL can be an expensive reagent in a lab, and what with it being involved in the final stage of a western blot, it’s something you don’t want to have to worry about too much. During my PhD, I was struggling with my western blots for ages—it seemed I was doing everything right, I just wasn’t getting any signal in the darkroom. At the time I was using a pre-made ECL and a friend that I shared lab space with suggested I try her homemade brew. I was sceptical at first, but later I came out of the darkroom with the biggest grin on my face—I had an epic signal on my film! Since then, my lab has used this simple, and very cheap, homemade recipe, and we haven’t looked back once.
Why not make your own ECL in the lab? The cost (if you assume that you already have a supply of hydrogen peroxide available to you) is roughly £50 ($70), which is a lot cheaper than many pre-made kits, which can cost hundreds of dollars/pounds. Having all the reagents available to you in the lab also means that you can easily make more up if you’re concerned that your stock has gone off—no need to wait for another order to come through!
You’ll need:
- Luminol (made up to 250mM in DMSO, store in the dark at -20°C). Available from Sigma Aldrich from £47.10/$72.90.
- p-Coumaric Acid (made up to 90mM in DMSO, store in the dark at -20°C). Available from Sigma Aldrich from £6.72/$10.40.
- 30% hydrogen peroxide
- 1M Tris-HCl pH 8.5
- Distilled water
Method:
Take two containers (they need to be something with a secure lid and which you can keep out of the light) and label one ‘A’ and one ‘B’.
In the first tube (A), combine the following:
- 1 ml luminol solution
- 0.44 ml coumaric acid solution
- 10 ml Tris-HCl 1M, pH 8.5
- Distilled water up to a final volume of 100 ml
In the second tube (B), combine the following:
- 64 µl hydrogen peroxide
- 10 ml Tris-HCl 1M, pH 8.5
- Distilled water up to a final volume of 100 ml
STORAGE: Keep both solutions in the dark at 4°C. I used to wrap my solutions in aluminium foil to keep the light away from them or keep them in a dark-coloured glass flask.
SHELF LIFE: The solution generally lasts up to 1-2 months if stored correctly. If you’re concerned that it’s been sitting in the fridge for too long, it’s probably best to just make more.
NOTE: You can easily halve or quarter the volumes of solutions described here if you want less than 100 ml each of solutions A and B—if your lab only does western blots sporadically, this may be useful to you, as it’ll waste less solution if it goes off in between experiments.
To use:
This homemade ECL is used in just the same way as a pre-made one. Combine equal volumes of each solution in a separate tube and mix. Cover your blot with the solution and leave for 1 minute, then drain off the fluid and expose it to your film.
Easy peasy! Hopefully, this quick, simple and cheap solution will be of help to you!
For more tips, tricks, and hacks for getting your experiments done, check out the Bitesize Bio DIY in the Lab Hub.
Originally published June 29, 2012. Updated and revised July 13, 2015.