Chemiluminescence, western blotting, problems...

Giampaolo Minetti minetti at sage.cc.purdue.edu
Tue Mar 5 11:58:54 EST 1996


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.
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 
happened.
The washing buffer is a Tris/HCl ph 7.5, 150 mM NaCl, 0.05% Tween 20.

What I know for sure is that the horseradish-luminol reaction is prone to
artifactual results. The presence of heme or hemoglobin affects the result,
because of a peroxidase-like activity of hemoglobin. (but in this case 
I don't have any hemoglobin in my samples!). Another amazing
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.

I feel that my problem is a contamination in one of the following:
buffers, electrophoresis apparatus, blotting apparatus, BSA and, last
but not least, DEIONIZED WATER
As for the nature of this contaminant... no idea. (actually I'm strongly
tempted to consider Fe contamination in my deionized H2O ?!?)

If anyone else ran into the same problem, please help.
Thank you very much for you attention





Giampaolo


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                                                               ~.~.~.~
                                                                ~.~.~:~
                                             Giampaolo Minetti     ~.:~
                                             Purdue University      \:/
                                          Department of Chemistry   /:\
                                          327  Wetherill Building  /:.~\
                                          West Lafayette IN-47906 /-~_~_\
                                             Tel. 317-494-5275   (_______)
                                      ****************************^*^*^*^
					minetti at sage.cc.purdue.edu



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