yeast one hybrid system
svetlov at oncology.wisc.edu
Wed Nov 11 17:34:22 EST 1998
In article <01be0cb7$4fa1b9e0$d263d680 at bootp.virusoppi>, "John Brunstein"
<john.brunstein at helsinki.fi> wrote:
> Generally, as you point out, a one-hybrid screen is used to find
> gene encoding an unknown protein binding to a known DNA sequence, or just
> the reverse of what you are trying to do.
> A more direct approach which has been successful in at least a
> papers as I recall was to use your known protein to gelshift a collection
> of oligo 'randomers' which have flanking defined sequences. You mix
> randomers and your protein, localize protein on the gel, isolate it.. and
> then PCR amplify the shifted randomers by way of their flanking defined
> sequences. If I recall correctly this process was repeated for three
> enrichment cycles before the amplifed product(s) were analyzed by
That works well enough and can be simplified further by making protein
his-tagged and instead of gel-shift using the affinity chromatography to
pull-out the oligos that bind to the protein of interest. Some people tell
me they PCR the Ni-NTA beads directly with proteins and DNA on them but
this maybe too much. There is actually more than a couple of papers that
successfully utilize one or another variation of this approach to determine
the consensus of a binding site, sometimes providing additional insights
not obvious from analysis of the genomic binding sites.
Vladimir Svetlov, PhD
McArdle Lab for Cancer Res.
1400 University Ave.
Madison, WI 53706
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