transcriptionfactor analysis

Mike Smith mfs3k at virginia.edu
Thu Jul 9 08:33:13 EST 1998


Frederik Boernke wrote in message
<35A536EC.72CB at nospam.ipk-gatersleben.de>...
>Dear friends,
>
>if have isolated this putativ DNA binding protein in a two-hybrid
>screening and it passed all the control transformations, so I tend
>to believe it's true. So my question is: Is there a way to identify
>the sequences/motives this protein may bind to? If it was the other
>round, identification of proteins binding to a certain DNA,
>south-western
>or one-hybrid would probably be the methods of choice. What about
>reversed one-hybrid/west-southern (ha, ha!), are there any protocols
>existing?
>Any hints will be appreciated.


The method you are looking for has been well described.  Check out these
refs:
Mavrothalassitis et al. 1990. DNA Cell Biol. 9:783-788
Pollock And Treisman 1990.  Nucleic acids Res. 18:6197-6204
Kunsch et al 1992.  Mol. Cell.. Biol. 12:4412-4421.

Basically you do a standard gel shift reaction using a random
oligonucleotide that is flanked on either side by a known sequence so you
can subsequently PCR and clone it.  You do the gel shift, elute the shifted
complex, amplify by PCR, and clone.  What you'll end up with is a series of
sequences from which you should be able to derive a consensus.

Haven't actually tried it myself so I can't make any claims as to how easy
it actually is to do.  And I'm sure it's got some cute name and acronym to
go with it.  Maybe somebody else knows?

Good luck.
Mike Smith, PhD
Univ.. of Virginia







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