Chemiluminescence, western blotting, problems...

John K. Troyer, PhD. jtroyer at
Tue Mar 5 18:31:08 EST 1996

On 5 Mar 1996, Giampaolo Minetti wrote:

> 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.



While contamination of one of your buffers may be causing your background
problems, may I suggest a technique for handling your blots following the
addition of ECL reagents:   You mentioned that you cover the membrane
with saran wrap.  A better method is to add your ECL reagents to the blot
and incubate for 1 min. then pick up the membrane at a corner with a pair
of forcepts and let as much of the liquid drip off as possible (blotting
the corner on paper toweling helps to speed this step).  Once you have
removed as much of the liquid as possible, place the membrane between
two sheets of transparency film or a plastic report cover and, using a 
kim-wipe, squeegy out any remaining reagent and air bubbles from the top 
of the membrane.  These steps are important because if you have excess ECL 
reagent on the top of your blot it can be activated and will spread out 
over the entire surface of the membrane.  Many times, this type of 
background looks swirly and liquid-like.  You may also be able to 
reduce the amount of background if you reduce the amount of sample you 
load onto your gel.  The ideal is to only activate the ECL reagents at 
the sight of a band.  If there is too much reagent or if your signal is 
too strong, the activation can spread over the entire membrane.

> observation of mine is that if you have carbonic anhydrase in your gel
> (as often happens because C.A. is commonly used as a molecular weight marker
> in most of the commercial preparations) and you transfer the protein on
> nitrocellulose and you soak your nitrocellulose in the Chemiluminescence
> reagent (WITHOUT any primary or secondary antibody) and you expose the film,
> you'll end up with a wonderful band of 29-30 kDa.

This effect can be very beneficial since you can then exactly determine 
the position of your bands relative to the mw markers on a Western blot!!

hope this helps.


John K. Troyer, PhD.
University of Maryland School of Medicine
Department of Biological Chemistry
jtroyer at

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