Black membranes

[Dr Engelbert Buxbaum]

Amanda Beardsley 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?

Sounds like your primary antibody is way to concentrated. Try a 10-fold
dilution and see what happens (you may need to dilute even more). Other
option: If your signal is so strong you can do staining directly on the
blot with diaminobenzidine/nickel:

20 x HRP-substrate: 6.01 g Tris (1M final), 14.6g NaCl (3.2M final),
950mg NiCl2 x 6 H2O (80mM final, CoCl2 may also be used) dissolved in 40
ml water. Adjust to pH 7.6 with HCl, and add 250 mg DAB (13 mM final).
Dilute to 50 ml with water, filter and freeze in 1 ml aliquots. Stable
for years at -20 C. When needed, thaw 1 of the vials, dilute to 20 ml
and add 1 ul 30% H2O2. Incubate the blot in this solution until bands
are satisfactory, then rinse with tab water and air dry. The black
precipitate of Ni formed scans and photographs well and is stable for
years in a note book. Careful with the DAB though, it is a suspected
carcinogen. You can buy it as tabletts (e.g. from Fluka) to make
handling safer.