Non specific carrier DNA in gel shift assays.

[Antonin Tutter]

>A few weeks ago I posted a message concerning gel shift bands.  I have
>tried the various suggestions and was able to get a very prominent band
>using salt and non-ionic detergent in the absence of poly di/dc.  I have
>since added the poly di/dc and found that this band dissappears.  My
>concern is when using this non-specific carrier DNA, how is it able to
>remove such a prominent band(that seems to be specific).  It seems as
>though the carrier DNA may be affecting my assays.  Thank You
>
>Ezra Schildkraut
>University of New Mexico
>

I remember posting a reply to your original question.  poly(dIdC) is a
non-specific competitor, however if the band you suspect to be specific is a
major-groove-binding protein, the dIdC may be outcompeting your probe.  dIdC
generally mimicks a major groove like GC-rich sequences do.  Try switching
to poly(dAdT) and see if that also outcompetes your probe.  If so, your band
is probably not specific.  


good luck,


_______________________________________
Antonin Tutter
Salk Institute for Biological Studies
RBIO-J
10010 N. Torrey Pines Rd.
La Jolla, CA  92037
email:  atutter at aim.salk.edu
web:  http://www-biology.ucsd.edu/~atutter/