Home made ECL CHEAP, QUICK RELIABLE

Dear HRP detectors,

In the early years of ECL when the kits weren't always reliable we developed the following protocol that gave excellent results at a ridiculously low price. It mainly relies on absolute purity of the p iodophenol and the luminol and probably needs the help of a friendly chemist. Ask the chemist to sublime the cheapest p-iodophenol you can find at 85C in vacuum. An ancient, somewhat forgotten technique, but extremely easy and it gives a perfect product. A mucky yellow powder giving beautiful white crystals.

Suspend 3g of cheap luminol in 90ml of butanol-1 and add 40ml of 1M NaOH to dissolve the luminol by warming and agitating. Add 30ml methanol, cool the recipient in ice and add 40ml of 1M HCl. Leave at 20C for 30min. Filtre the crystals on a Whatman no. 1 filtre and rince with dichloromethane. Suck air through to dry, preferably protected from the light with a piece of foil.

Take all the usual precautions during these simple manipulations. You can stock the two products in a dessicator, the luminol in a bottle wrapped in aluminium foil to protect it from the light.

Two solutions are made up. Solution 1 contains 780mg luminol and 950mg p-iodophenol in 1litre 0.1M tris-HCl pH 9.35. Solution 2 contains 100 microlitre 30% H2O2 in 1litre 0.1M tris-HCl pH 9.35. Both solutions are stable for several months at 4C in the dark (in the fridge). Just before use, mix 1 volume of each solution.

As the adverts say, you'll be amazed by the results.

Happy if this helps,

David

> Company kits end up costing $3-5 per western blot which is 90% of the

> cost of the whole procedure.

>

> People must have some good homemade HRP (and AlkPhos) Chemiluminescent

> Detection protocols or references?

>

> Could you send me one or point me to it? Thanks.

*Luminol 4mg/ml in DMSO	1ml
*p-iodophenol 1mg/ml in DMSO	1ml
1M Tris.HCL pH 7.5	0.6ml
H2O2 (30%)	5 uL
H2O	7.5ml

*Store at -20

Mix this stuff up fresh a few minutes before use.

Works for me!

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