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?
> 
> 
> 
> 
HI,
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

Toumy




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