Black membranes

Pow Joshi via methods%40net.bio.net (by pow.joshi At gmail.com)
Tue Feb 6 11:21:23 EST 2007


On 2/4/07, Amanda Beardsley <amanda.beardsley At gmail.com> wrote:
> 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?

I have about three questions/suggestions:

is your Goat anti-mouse HRP rather old? .... you could try changing
the secondary to a newer one..... I can only think of the secondary
being degraded and giving you a non-specific background .....also, I
generally use about 1:8000 or 1:10000 dilution for secondaries.

secondly, like Tom suggested, you could try a different blocking
buffer (with tween 20 if you are'nt already using it) and use 5% BSA
instead of non fat milk or vice versa.

Thirdly, you could try changing the membrane ....

hope that helps
pow

>
> Any other ideas would be most appreciated.
>
> Amanda :)
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