anti-HA Western / Immunoblot nonspecific bands

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

"Andrew" <aji23 at> wrote in message
news:958c94ef.0401201150.5a257d69 at
> 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

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