homemade ECL

[allisonh]

While digging through some old papers I found a newsgroup posting with a
recipe for ECL reagents (posting was from 1998 so practically the dark
ages in internet years). Is anyone using homemade ECL currently? I am
wondering if it is worth my time to order ingredients and try it out or
will I be disappointed by the sensitivity.
TIA
Allison

[Jose de las Heras]

"DK" <d...@no.email.thankstospam.net> wrote in message
news:6n2Aj.486$9b1...@newsfe06.lga...
> In article <47d04ed4$0$16009$9a6e...@news.newshosting.com>, allisonh

> <all...@nospam.com> wrote:
>
>>Is anyone using homemade ECL currently? I am
>>wondering if it is worth my time to order ingredients and try it out or
>>will I be disappointed by the sensitivity.
>

> I remember trying it at around this time and comparing it back to
> back with Pierce's "SuperSignal" and concluding that, for as
> long as I can't make home-made Pierce version, the trouble of
> making traditional ECL is not worth it if you want sensitivity and
> peace of mind every time you do Western where you don't
> exactly know what kind of signal to expect.
>
> Truth is, however, I was never satisfied with Amersham's original
> ECL mix either.
>
> DK

We've been using a homemade version for years now. Pretty cheap and easy.
Out of my head, I just add 670ul of 1.5M Tris pH8.8 and water to 10ml. Then
we have frozen aliquots of luminol and p-coumaric acid (i forget the
amounts/concentrations... can you tell I just do it mechanically now?)...
all we do is add one aliquot of each, mix well, add 3-4ul of H2O2, and pour
over the membrane.

I don't do a lot of westerns, but others in my lab do and noone complained
about sensitivity when we stopped buying the commercial version. The one
thing that is different, is that our homemade version goes off a lot more
quickly. I think that after 15 minutes or so you won't get much. But often
we only need to expose for a minute or so to get good signals (of course,
thsi is highly dependant on the antibody and what you're actually after)...
My "routine" tends to be a quick exposure (20-30 seconds) and expose another
film while developing the first. It takes about 2min for the film to come
out. Then depending on what i see, either I develop the one I've been
exposing (2min already) or leave it a bit longer. You do have to work a bit
faster than with commercial ECL, but it works quite well once you get the
hang of it.

If interested I can post our recipe. The ingredients are cheap and they'll
last a looong time.

Jose

[Aawara Chowdhury]

In <63egs3F...@mid.individual.net>,

Jose de las Heras <jos...@tiscali.co.uk> wrote:

> I don't do a lot of westerns, but others in my lab do and noone complained
> about sensitivity when we stopped buying the commercial version.

The Pierce Supersignal reagents that Dima referred to are far more sensitive
than Amersham's ECL (ECL plus) reagents. We also use a home-made ECL
reagent that is similar to yours, and the sensitivity of our reagent is
similar to Amersham's ECL reagent (or Pierce's ECL reagent).

The Supersignal reagents permit detection of 10 - 50 femtograms of protein.

In practical terms this means that if we use a particular antibody at a
1:1000 dilution with our home-made ECL reagent, we can use it at 1:15000
with the Supersignal Femto reagent. Useful for some Abs that are very
expensive.

AC
--
Email: echo 36434455860060025978157675027927670979097959886449930P | dc

[ar...@bio.umass.edu]

I regularly do western blots with a method similar to the one described by
Jose. It works well for standard western blots. In my experience it is
not as sensitive as commercially available reagents such as Pierce Super
Signal West Dura. This is sometimes a benefit however, b/c it may result
in less background.

If I'm working with a new antibody or sample type, I usually use the
homemade reagents, and if greater sensitivity is required I move on to the
commercial ($$$$$$$$) stuff.

Stock sol'ns (50 ul aliquots)
1)90 mM coumaric acid
2)250 mM luminol

Working sol'ns
A) Add 22ul coumaric acid and 50ul luminol to 5mL cold 0.1M Tris pH8.5
B) Add 3ul H2O2 to 5mL cold 0.1M Tris pH8.5

Mix A) and B), add to blot for 1 min, continue with exposure to film etc.

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[allisonh]

I'm using ECL plus right now and find that the sensitivity is fine for
what I am doing. So I would be interested in trying a homemade version
to save $$.
One question though - since it is only active for 15 minutes, is it
possible to 'reactivate' the blot by resoaking it in the reagent if the
correct exposure is not done the first time thru? I'm thinking of the
days when there is a lineup in the dark room or the like :)

Also, could you please post your recipe (if it is different than the one
that Arnec posted).

Thanks a lot
Allison

[ar...@bio.umass.edu]

If you need to redo a detection, I rinse the blot in PBS, then put it back
into secondary (which I would have saved) and continue from there.

Also, in the protocol that I summarized previously, I the coumeric acid
and luminol are dissolved in DMSO.

Arne