homemade ECL

[allisonh]

Jose de las Heras wrote:
> "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 
> 
> 
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