homemade ECL

[arnec from 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.



>
> "DK" <dk from no.email.thankstospam.net> wrote in message
> news:6n2Aj.486$9b1.425 from newsfe06.lga...
>> In article <47d04ed4$0$16009$9a6e19ea from news.newshosting.com>, allisonh
>> <allison from 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
>
>
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