Black ECL blots

Annette C. Hollmann ah690549 at bcm.tmc.edu
Tue Apr 4 17:29:02 EST 1995


In article <3lru8d$pmk 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

I have had similar problems when blotting with polyclonals.
The only 2 ways around the problem that I found are:

1. Use a monoclonal antibody.

or

2. Partially purify the protein first (I use immunoprecipitation, but
column chromatography may also work). Also, titrate the antibody way down
(like in the 1:4000 to 1:40000 range)

Even when using a monoclonal antibody, you might still run into problems
with some samples because the ECL system is so darned sensitive.
It helps not to load too much protein and to titrate the antibody as low
as it will go.
If you get an image after a 1 second exposure (or you can see the blot
glowing in the dark), you can get away with using less antibody.
If you need 15 to 30 seconds to get a decent image you are probably in the
ballpark.

I hope this helps


Annette
ah690549 at mbcr.bcm.tmc.edu




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