Black ECL blots

John A. Newitt newitt at
Tue Apr 4 18:03:05 EST 1995

In article <3lru8d$pmk at>, jcim at
(James Stiehr) wrote:

> 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. 
I've never had this problem.  Have you used the primary antibody with this
blocking agent and an alternative detection system before with success? 
Could it be possible that your primary antibody is reacting with something
present in your blocking agent?  For example, say you blocked with buffer
containing nonfat dry milk to block, but your antiserum reacts with a
protein that is present in milk.  This could be due to either a relevant
reaction, say galactosyl transferase antibody reacting with GT that gets
secreted in milk, or an irrelevant reaction, the immunized animal produces
antibodies that react with something in milk unrelated to the antigen. 
Switching to a different blocking agent may alleviate the problem for both
of these situations.  Alternatively, for the second situation, you could
keep the same blocking agent, but affinity-purify the primary antibody
prior to blotting.

John A. Newitt  <newitt at>
National Institutes of Health
Bethesda, Maryland  USA

More information about the Bioforum mailing list