Hi Simone
If your band appears when you aren't using any antibody for the IP,
then it sounds like your background band is due to some non-specific
binding (in theory, you shouldn't be able to collect any protein if
you don't add antibody during your IP). I had (and still have, by the
way) a problem with protein g falling off the sepharose beads I was
using (about 30kD). The band protein g formed, fell directly over the
top of the protein I was looking for, and it bound any secondary
antibody I used for the western (does the band appear when you just
incubate a western blot from your IP samples with only a secondary
antibody? Protein G binds all antibodies..). I'm not suggesting this
is your problem, but in your case, in theory when you do a
densitometry analysis of your blots, you should be able to subtract
the background from your test bands. Maybe your band arises from
something binding the sepharose beads directly, especially if you
aren't preclearing your lysate or if the final washing of the beads is
insufficient..
just a thought!
Cheers,
Melissa
