ECL Western troubles

Toumy Guettouche tguettou at newssun
Tue Sep 3 14:22:07 EST 1996

On 2 Sep 1996, Jose Bonner wrote:

> We're having trouble with Western blots probed by ECL.  Films sometimes 
> come up blank, even though antibodies and reagents are OK.  Sometimes a 
> 5-minute exposure is fine, but a 1-hour exposure (started just after) is 
> blank--not even any background.  What can interfere with ECL?  The 
> problem comes and goes; good blots interspersed with blanks.  Reprobe 
> the blanks, and sometimes they come up good; reprobe the good ones, 
> sometimes they come up blank.  Anyone else have this problem?
we had and sometimes still have the same problem with our ECL system. One 
problem was when we switched from Dupont's ECL system to 
Pierce'ECL-system. You have to dilute the secondary antibody much higher 
(1:10-50000 vs. 1:2000), otherwise you apparently shift the reaction from 
a long lasting glow to a short flash of light. They just mentioned that 
in their new, not in their old protocolls. You usually have a problem 
if you can see your bands glowing strong in the dark room when the red 
lights are out.
Hope that helps


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