ECL Western troubles

[Peter Heifetz]

In article <50fpck$f9n at dismay.ucs.indiana.edu>, Jose Bonner
<jbonner at bio.indiana.edu> 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?

I had a similar sporadic ECL problem, using Dupont reagents: strong signal
at 2-5 min, which rapidly (and completely) disappeared over 30 minutes or
so. The manufacturer was stumped as well.  I ultimately "solved" the
problem by rinsing all glassware (pans, roller bottles, etc.) with concn
HCl followed by much water prior to each experiment.  I never found out
what was quenching the luminescence  but it did not recur with the
acid-washing.

cheers,

-- 
Peter Heifetz
Duke University
Developmental, Cell, and Molecular Biology Group