ECL Reagents

[Wolfgang Schechinger]

Hi Zac, here it is. A whole bathtube of ECL mix for the price of one 
kit:

Solution 1: (prepare as much ECL as you will use in 1-2 month)

780mg/l (4.4mM) Luminol, 950mg/l (4.3mM) p-iodophenol (pIP), or 
(cheaper, subjectively a bit better/more intense light) 4.3mM 
4-methyl-umbelliferon (MUBF, M=194.18, calculate the amount 
yourself)

prepare 1M stock in DMSO of each compound (store dark in the fridge 
or at -20), 

dilute with 0.1M Tris-HCl pH 9.35, store dark in the fridge, too

Solution 2: 400µl/l 30% hydrogen peroxide in the same buffer

Solution 2 is stable for 2-3 month in the fridge; I regenerate it 
then simply by adding H2O2.

Test procedure/quality control: 

Place equal amounts (say 100ul) of Soln 1 and Soln 2 in an eppi, put 
1 ul HRP-conjugated AB in the lid, go to a dark place, close the eppi 
and invert. You should *immedeately* see the bright glow.

For a 10x20 cm membrane, I use 3ml of each solution. I mix 
them in dim light directly on the membrane. Be sure not to 
cross-contaminate the solutions.

I currently use Luminol and MUBF from Fluka without further 
purification. The pIP I tested was from Lancaster Synthesis and 
worked ok, too.

References: 
JBC article in 1992 (vol 267 pp 21830-21838).
I got the receipe from David.SHIRE at TLS1.elfsanofi.fr

Enjoy!

Wolfgang


> A few months ago, I saw several posts about making ECL reagents from
> commercially available sources of Luminol (3-aminophthalhydrazide)
> and 4-iodophenol. Some individuals gave the concentrations needed,
> and even the catalog numbers of some of these items. Does anyone
> have such info or is there a www site which contains it?
> 
> Zac Pujic
> dnzpujic at mailbox.uq.edu.au
> Department of Dentistry
> The University of Queensland
> Queensland, Australia
> 
> 
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usual disclaimers apply * This message is RNAse free - please don't touch!
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Wolfgang Schechinger         
University of Tuebingen, Germany
email: wgschech at med.uni-tuebingen.de * wwWait: http://www.medizin.uni-tuebingen.de/~wgschech/start.htm
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