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high background after ECL exposition - (Jan/10/2011 )

I got high signal on my W.B. with background. with some saturated lanes.
i tried to scan again the filter on the storm, without using ECL Plus again, so, i could see a little bit better signal.
saturation still present. i'm trying to block again the filter and scan on the storm without any other incubation with antibody or ECl exposition!
do you think is it correct to do it??


If the membrane (I presume this is what you are calling the filter - it will be either nitrocellulose or PVDF) has been exposed to antibody, re-blocking probably won't make much difference as the antibody will be bound to the "unblocked" parts of the membrane so you will still get lots of signal.


Sometimes I find useful killing the peroxidase (put the membrane on parafilm, cover with some drops of 30% H2O2, incubate 15 min at 37 C, wash the H2O2 and reblock 15 minutes). By doing so, all your antibodies remains attached to the membrane but the HRP is killed and will never bright again.

From this point, I do again the secondary antibody incubation, washings and ECL. The fresh secondary, still recognizes some primary but the background now is less (as saturated by previous silent antibodies).

Sometimes works, sometimes not.


Sometimes I get very high signal too specially for my GAPDH blots. What we do is use Versadoc (not films) and use an exposure time of 0.1 sec and acquire the image. The blots come out pretty well whereas they would have been saturated by few seconds exposure. I hope this helps.