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how to make your own ecl so your western blots actually work
bio wetlab
Western blots are the workhorse of modern molecular biology. These days we use enhanced chemiluminescence (ECL) because it is safer than radiation. However, commercial ECL reagents are expensive. Your labmates may also not handle your supply of the light-sensitive ECL reagent as carefully as you would like. This can result in frustration and days of effort wasted on failed blots.
You can mix up your own ECL reagent to guarantee purity to avoid the above.
what is in an ecl reagent?
There are four ingredients:
- A buffer: Tris-HCl
- An oxidiser: hydrogen peroxide
- Luminol
- A chemiluminescence enchancer
As you probably know, luminol reacts with an oxidizing agent in the presence of a catalyst to produce excited 3-aminophthalate dianion. The relaxation of the dianion results in a brief and faint flash of 425 nm light. Because this is so brief and faint, we need to amplify and buffer the photons so we can actually measure them. This is the purpose of the enhancer.
You have many choices of enhancer:
- p-Coumaric acid (pCA)
- 4-iodophenylboronic acid (4IPBA)
- 4-iodophenol (4IP)
Personally I agree with Hann and Behrmann that 4IPBA tends to work better. See their paper referenced below. I have never used 4IP and cannot comment on that.
recipe
With the stock solutions I use:
Solution A (10 mL final volume):
- 1 mL of 1 M Tris/HCl pH 8.8
- 50 µL of 250 mM luminol
- 22 µL of 90 mM pCA or 222 µL 90 mM 4IPBA (depending on preference)
- ~9 mL of water (make up to volume)
Solution B (1 mL final volume):
- 900 µL of water
- 100 µL of 30% hydrogen peroxide
Store in the dark and at 4°C before use. Do not keep for too long, preferrably use immediately. Before use, mix 1 mL of solution A with 3 µL (for pCA) or 6 µL (for 4IPBA) of solution B. You will need about 100 µL of mixed solution per square centimeter.
Experiment and adjust concentrations to suit your own lab and experimental conditions. You may prefer using DMSO.
disclaimer
Disclaimer: Use this information at your own risk. Just use the commercial stuff if you don't want to homebrew. This information is provided with no warranty of any kind and may not be applicable, appropriate, or safe to your situation. I am not liable for any claim, damages or other liablity from your use or other dealings with this information. Do not just follow recipes on the internet without doing your own research first.
references
- Haan C, Behrmann I (2007) A cost effective non-commercial ECL-solution for Western blot detections yielding strong signals and low background. J Immunol Methods 318:11–9. doi: https://doi.org/10.1016/j.jim.2006.07.027
