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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 dark room. At the time I was using a pre-made ECL, and a friend that I shared lab space with suggested I try her home-made brew. I was sceptical at first, but later I came out of the dark room 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, home-made recipe, and we haven’t looked back once.

The cost of making your own ECL in the lab (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 from £43.10/$63.20.
  • p-Coumaric Acid (made up to 90mM in DMSO, store in the dark at -20°C). Available from Sigma from £5.70/$8.40.
  • 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 home-made 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 to your film.

Easy peasey! Hopefully this quick, simple and cheap solution will be of help to you!

4 Comments

  1. memari on September 25, 2013 at 10:12 pm

    Hello
    How to make it very sensitive to detect Femto gram of Protein?

    Thanks
    Babak

    • Maibritt on July 13, 2018 at 10:54 am

      I World also like to know the anser for Tvis question. Companies are Boe maling Extracellular sensistive ECL for weakly expressed proteins, and I bet it just requires changing simple parameters of this recipe. Does anyone know what parameters to change and how? Please answer :-). Kind regards, frustrated postdoc #1973

  2. Harley King on June 29, 2012 at 12:38 pm

    Great recipe, Steffi. You’re right, using homemade ECL can save a lab a lot of money and is just as good as the store-bought stuff.

    • Steffi Magub on July 2, 2012 at 12:44 pm

      Thanks! It seems silly to spend so much on it when it’s so easy and cheap to make your own solutions!

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