Help-Request: Unspecific Signal by Sekundary AB in Western Blot

Thomas Seib hgtsei at med-rz.uni-sb.de
Tue Feb 10 06:22:53 EST 1998


Non-specific bands produced by secondary AB are not unusual. In my
hands, using the Amersham anti-mouse IgG, it was a matter of dilution
to get rid of these bands (1:5000). Did you try that?
Matthias



In article <6bp5pb$ick2 at janus.gsf.de>, rosemann at gsf.de wrote:

> We have tried to get Western Blots for P53 and Bax working and encountered
> the following problem: The secondary antibody (Amersham's rabbit anti-mouse
> IgG, HRP conjugated) alone produced a lot of unspecific bands,especially
a very 
> prominent one at approx. 56kD. We already tried to get rid of it by mixing
> the secondary antibody with the protein extract or with BSA, but with
little or
> no success. 
> 
> 
> Even more surprising was the observation, that another secondary antibody
> (Santa Cruz' Donkey anti-goat IgG, HRP conjugated) produced an
unspecific signal at the
> same position on the blot. 
> The unspecific band on the Western appeared with protein extracted from
> rat cells as well as from human cells, but not from NIH 3T3 mouse cells. 
> We could justify that no endogenous peroxidases are responsible for this
> effect and that none of the reagents were contaminated. 
> For the detection we use Amersham's ECL system.
> 
> 
> Somebody proposed, that the Bromphenolblue containing loading buffer
> (Laemmli's buffer with DTT) might be the reason, since other people
using a recepie with
>  Coomassie blue containing loading buffer (+ Thioglycolic Acid instead of DTT)
>  told me that they could get rid of unspecific signals, however
> they could not tell me whether they came from the first or the secondary
Ab.    
> 
> 
> I would like to hear from anybody who can give advise or simply tell me of
> similar observations.
> 
> 
> 
> 
>         Thanks in advance       Michael     (rosemann at gsf.de)
>

-- 
Thomas Seib
Kantstr. 12
66292 Riegelsberg



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