Black membranes

Amanda Beardsley via (by amanda.beardsley At
Sun Feb 4 19:42:00 EST 2007

Hi all,

I'm having a little trouble with one of my Western Blots. I'm using a goat
anti-mouse-HRP secondary antibody against my mouse monoclonal (against Lis1
protein) at a concentration of at least 0.02ug/ml (each western I do I'm
diluting even further), and I'm still getting a black western. If I hold the
film over the blot for an instant (what we call a flash exposure) my bands
are visible upon development, but the background is so heavy that I'm not
able to scan the film and still see the bands. I incubate the ECL reagent
for 2 mins and blot it dry with filter paper before exposing it. This is
only happening with this antibody (however I only have one mouse antibody
that I'm using at the moment). I also use a goat anti-rabbit at a much
higher concentration with no trouble. Apart from diluting even further, the
only other thing I can think of is if anyone ever rinses the membrane in
buffer before exposing it to film?

Any other ideas would be most appreciated.

Amanda :)

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