Horrendous background problem on chemiluminescent westerns

[Dr. Peter Gegenheimer]

On Wed, 9 Sep 1998 09:03:01, "Frank O. Fackelmayer"
<fof1 at chclu.chemie.uni-konstanz.de> wrote:

> Try using your antibodies, both primary and secondary, at a much higher
> dilution. Heavy background is mostly the result of too much antibody. As a
> rule of thumb, use 1/10 the amount of primary antibody (in comparison to
> colorimetric detection), and the lowest possible amount of secondary ab. In
> our hands, the POX-coupled secondaries from sigma, diluted 1:100.000 (that‹s
> NOT a typo) in new blocking solution (TNT with 5% dry milk), works great. You
> might have to titrate both antibodies, but it is really worth the work.
>
> Frank
>
>
>
> Guess wrote:
> >
> > Hi.  I am hoping somebody can give some tips on cutting down the
> > background on my chemiluminescent westerns (ECL reagent).  I get large
> > spots, patches and smears all over (as opposed to a nonspecific
> > recognition/banding pattern).  Its particularly bad  where the edges
> > of the gel contact the membrane during transfer.

I second the antibody dilutions!! We had the same experience with the Pierce
SuperSignal chemi. system: the film came out completely black. After we
titrated the primary & secondary Abs (like a good immunologist would have
done), we found that 1) primary Ab which was used at 1/2000 to 1/10,000 for
colorimetric detection worked best at 1/50,000 to 100,000 (1e-5, no typo); 2)
secondary HRP-conjugated Ab, which Pierce recommends using at (I think)
1/20,000 - 1/50,000, worked best at 1/50,000 to 1/100,000. Also 3) blocking at
room temp for 5 to 10 hr in PBS-BSA-Tween (0.1%, I think); 4) washes after 2nd
Ab were 2 X 5-10 min in PBS-BSA (no Tween), then about 4X in PBS-BSA-Tween.

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