Black membranes

Tom Anderson via methods%40net.bio.net (by ucgatan At ucl.ac.uk)
Mon Feb 5 07:54:26 EST 2007


On Mon, 5 Feb 2007, Amanda Beardsley wrote:

> 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.

This sounds to me like it might be a problem with the primary rather than
the secondary. Can you scrounge a bit of mouse-anti-something from another
lab to see if that works?

If it is the primary, you could try diluting it down. And whether it's the
primary or secondary, two things you might try would be using a carbonate
rather than tris-glycine transfer buffer (sounds crazy, but this has
worked really well for me - 13 mM carbonate pH 9.9) and changing the
blocking conditions (if you're using milk, try BSA, etc).

> 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've never needed to do that - or even to blot the membrane before
imaging; i just pick it up with tweezers and let it drip-dry for a few
seconds.

Sorry i can't be more use!

tom

-- 
Tom Anderson, MRC Laboratory for Molecular Cell Biology, UCL, London WC1E 6BT
(t) +44 (20) 76797264   (f) +44 (20) 76797805   (e) thomas.anderson At ucl.ac.uk



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