anti-HA Western / Immunoblot nonspecific bands

Tarja Kokkola tarja.kokkola at uku.fi
Thu Jan 22 02:07:25 EST 2004


"Jens" <tinajens at mail.dk> kirjoitti
viestissä:400ed8a1$0$280$edfadb0f at dread12.news.tele.dk...
>
> "Andrew" <aji23 at drexel.edu> wrote in message
> news:958c94ef.0401201150.5a257d69 at posting.google.com...
> > Hey All,
> >
> > I'm currently doing Western Blots on mammalian cell lysate (directly
> > lysed with SDS-PAGE sample buffer, run out on an 8% gel and
> > transferred to nitrocellulose).  I'm using the 12CA5 anti-HA antibody
> > available from Roche at a dilution of 1:1000 in PBS +  1% Milk / 10%
> > goat serum.  I am transfecting an HA-tagged protein into U373 cells; I
> > include an untransfected control.  My problem is this - the negative
> > control is showing a clear doublet running at ~85kDa.  It's a problem
> > because my protein of interest runs close to this as well.
> >
> > Has anyone seen these nonspecific bands before and removed them?  One
> > suggestion is to preabsorb the antibody against the negative control
> > lysate, but I was wondering if the problem has already been tackled by
> > someone.
> >
> > Thanks for your time.
> >
> > -Andrew
>
> Maybe not what you would like to hear, but the 12CA5 antibody did not work
> well in my hands, however, clone 16B12 works very well in immunoblotting
and
> immunofluorescence.
> JM
>
>
I have also seen the nonspecific band in my Western Blots with 12CA5
antibody, both with CHO and COS-7 cell membranes and lysates. In our system
it was a single band close to 75 kDa. We used 10% PAGE and transfer to
nitrocellulose. The blot was incubated overnight in the cold room in 1:1000
dilution of the anti-HA antibody in TBS-Tween (0.05% Tween) + 2.5% milk. As
our target protein was clearly smaller than the nonspecific band, we were
able to just ignore the extra bands when quantifying our blots. Excess of HA
blocking peptide removed both the specific and the nonspecific bands.

I noticed something about the nonspecific band that I cannot explain and I
would be glad if somebody could explain this phenomenon for me:
Once, after stripping my blot I reprobed it with exactly the same conditions
as the first time (i.e. with 12CA5 anti-HA antibody). If somebody wonders
why I did it: the chemiluminescence reaction had been very uneven and I
tried to get a nicer picture + quantify the bands. In this reprobed blot the
nonspecific bands had disappeared but the specific bands were all nice and
clear!! How can this be possible? The samples were all CHO cell membranes.

- Tarja





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