ECL detection kit

[Curt Ashendel]

On 5 May 1995 23:02:44 GMT, 
Blake Meyers  <ez044224 at bullwinkle.ucdavis.edu> wrote:

>I'm curious if anyone has figured out a way to stretch the life of 
>Amersham's ECL detection kit. I find that we have plenty of labelling 
>reagent, blocking reagent and somewhat less of the hybridization 
>solution, but we quickly burn through the detection reagent. 
>
>Does anyone know if you can either dilute the detection reagents, or even 
>better, what's in them so I can make my own?
>

For protein blots I just use less than the instructions indicate (0.8 ml 
each reagent for an 11 x 14 cm blot). Just smear the liquid around in the 
bag and be sure to remove all the wash solution before adding the ECL or it 
will be diluted.

Dilution is not a big problem and affects the intensity in a non-linar way, 
since both subtrates are well above their Km for the enzyme. Dilution 
probably has more affect on the time course of the peroxidase reaction, 
which is notoriously non-linear. However, I have not tried dilution of the 
reagent as a means of stretching it.

There was a post to this group on how to make your own reagents last 
september by Yasushi Okada. I saved it, so here is the quote:

********
Here is the recipe I use,

Stock solns.
a) Luminol stock: Luminol (Sigma) 4mg/ml in DMSO.
b) Enhancer stock: p-iodophenol (Aldrich) 1mg/ml DMSO.
c) Tris Buffer: 100mM Tris.HCl pH7.5
d) H2O2: 3% H2O2

Stock solns a) and b) should be kept in -20deg for <6 months.

Working soln. (Prepare just before use)
a) 0.5ml, b) 0.5ml, c) 2.5ml, d) 25ul, ddw 1.5ml (final 5ml)

This is very cheap, and as sensitive as ECL kit.

Reference: Schneppenheim et al (1991) Electrophoresis 12:367-372.

Hope this helps.

--
Yasushi Okada, MD.
********



Curt Ashendel
Purdue University
West Lafayette, IN
ashendel at aclcb.purdue.edu