Chemiluminescence, western blotting, problems...

Giampaolo Minetti minetti at
Thu Mar 7 12:30:40 EST 1996

In article <4hhrse$ium at>,
Giampaolo Minetti <minetti at> wrote:
>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.
>I thought that I was using a too high concentration of secondary
>antibody (goat anti-rabbit,  horseradish peroxidase-conjugated BioRad)
>so I switched from a 1:10000 dilution to 1:30000. The only effect was
>a longer delay before the background luminescence showed up.
>The problem is the same, no matter what the primary antibody is, and no
>matter what solution (5% BSA or 5% non fat milk) I use for blocking the
>membrane before the primary. Also, I'm using a brand new Amersham ECL reagent.
>Since it looked like the secondary antibody was damaged, I tried another
>Goat-anti-rabbit-HRP (Pierce), with exactly the same result.
>There is some problem related to the secondary antibody. I've tried
>to soak a piece of plain nitrocellulose into the reagent, and nothing 
>The washing buffer is a Tris/HCl ph 7.5, 150 mM NaCl, 0.05% Tween 20.

I'd like to thank everybody for  your timely reply.
It turned out that the problem was due to the high concentration of 
the secondary antibody. I could not believe that I had to dilute the
secondary up to 1:100.000 (one to one hundred thousand), in order to
avoid the background. In these days I ran a lot of controls but none 
of them gave me the solution of the problem, except for the dilution
of the secondary.
Anyways, now I know the answer and I'll take benefit from this experience:
I had the same problems using PVDF membranes; I had the same problems no
matter what the primary antibody concentration was, I had the same 
problems no matter what the blocking buffer was.
At last I found that Enhanced Chemiluminescence is a lot more 
sensitive than I  imagined. (did they change the composition of the
reagent in the meanwhile?)


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