anti-HA Western / Immunoblot nonspecific bands

Andrew aji23 at drexel.edu
Tue Jan 20 14:50:50 EST 2004


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



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