Chemiluminescence, western blotting, problems...

Bernard Murray bernard at elsie.nci.nih.gov
Tue Mar 5 17:52:49 EST 1996


In article <4hhrse$ium at sage.cc.purdue.edu>, minetti at sage.cc.purdue.edu says...
>
>Hello,
>I am experiencing problems with Enhanced Chemiluminescence Detection
>(Amersham ECL) and western blotting.
>What happens is that after adding the ragent, incubating for 1 min and
>taking the nitrocellulose into the cassette for exposure (I use to
>cover the membrane with Saran Wrap), everything is OK.
>After 2 or 3 minutes, however, the entire membrane is luminescent. The
>bands I'm interested in are there, but you cannot take a good
>exposure because of the extremely high background.

<SNIP>

Have you tried reducing the amount of *primary* antibody?
You should drop the concentration considerably if you
are comparing between chloronaphthol etc. (colorimetric)
and ECL as substrates if you don't you will end up with
the result that you describe.
	You might also want to try another membrane as
PVDF etc. is supposed to have a lower background.
You can also reduce some of the background if you rinse
the substrate-exposed blot briefly in buffer (eg. PBS/
BSA/Tween) as this will cut doen the total output and
may let you see your specific bands.  There was also a
post a while ago that pointed out a Biotechniques article
that suggests diluting the ECL reagent before use.

		Bernard

Bernard Murray, Ph.D.
bernard at elsie.nci.nih.gov  (National Cancer Institute, NIH, Bethesda MD, USA)




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