Black ECL blots

[rjanssen_imm at opal.tufts.edu]

article <3lru7q$pku at pipe3.nyc.pipeline.com>, jcim at nyc.pipeline.com (James Stiehr) writes:
> Has anyone else ever had problems using polyclonal antisera on a western
> blot detected with ECL resulting in an overall black blot.  It is almost as
> if the blots were not blocked at all (they were).  The ECL supplier didn't
> have any suggestions.  If anyone has come across this problem and hopefully
> a solution, I would appreciate any suggestions. 
>  
> Regards  
> James Stiehr

Dear James,

Hm, this looks like a serious problem:

Assuming you did not expose the film to direct excessive light, the following
might be the case ?

- Excess of ECL detection solution on the blot. Be sure to drain all the
solution before you put the film on it.

- Do not move the blot while incubating with ECL detection solution. I have the
impression that moving will smudge your blot.

- Block extensively. I often experienced a lot of background staining which
only could be reduced by blocking the blot, before staining, with a 5% milk
solution overnight at 4 degrees C. Between 1st and 2nd antibody, wash blot 3
times with TBS/.05% tween. After 2nd antibody, wash 5 times with TBS/.05%
tween. Sometimes it is necessary to use .2% tween.

We do the following with "black films": Wash blot for 2-5 hours with TBS/tween
and incubate blot again with ECL detection solution. The black smudges were
gone and the bands were still visible.

Good Luck,

Richard Janssen
Hematology/Oncology, New England Medical Center
750 Washington Street, Boston MA 02111
email: rjanssen_imm at opal.tufts.edu