transcriptionfactor analysis

mbarnhart at my-dejanews.com mbarnhart at my-dejanews.com
Thu Jul 9 12:57:03 EST 1998


In article <6o2gmb$i9v$1 at murdoch.acc.Virginia.EDU>,
  "Mike Smith" <mfs3k at virginia.edu> wrote:
>
> 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
>

Since your gene is cloned.  Another option would be to select for bound
sequences using a GST fusion (or his tag, or whatever) and affinity
precipitate the DNA with glutathione beads or whatever.  The same PCR
amplification principle applies.  I think the technique is called SELEX if
memory serves.

Michael

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