wgschech at med.uni-tuebingen.de
Thu Jun 11 08:23:19 EST 1998
Hi Zac, here it is. A whole bathtube of ECL mix for the price of one
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
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.
JBC article in 1992 (vol 267 pp 21830-21838).
I got the receipe from David.SHIRE at TLS1.elfsanofi.fr
> 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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University of Tuebingen, Germany
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