anti-HA Western / Immunoblot nonspecific bands

Jens tinajens at mail.dk
Wed Jan 21 14:52:35 EST 2004


"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





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