William J. Meilandt (wmeiland at lonestar.jpl.utsa.edu) wrote:
: We have a putative DNA binding protein and would like to identify the
: genomic sequences to which the potein binds to. Does any one have
: references or ideas they would like to share?
: I am considering to purify the protein and put it on a column and then
: pass predigested genomic DNA thru the column to isolate genomic fragments
: which bind to my protein. Is it better to used protein purified from my
: tissue of interest or in vitro translated?
: An alternative approach would be to screen a genomic library with the
: putative binding site for the DNA-binding protein. Isolate this clone,
: digest it into smaller pieces, clone into a vector and re-screen with
: the binding site, and sequence the positive clones.
: Any suggestions on which method would work better, or if you know of a
: different method of isloating the targets of DNA-binding proteins,
: would greatly appreciated.
I know you did not specifically ask for criticism, but in my
opinion this may turn out to be a lot more work than you think and you
might reconsider whether it is really worth it. Having said that...and
in the spirit of helpfulness, I recall that several years ago, sometime
in the 80s, someone utilized a nitrocellulose filter-binding technique to
isolate a factor binding site that bound the transcription factor called
nuclear factor-1 (NF-1), which is a CAAT-box binding protein. You might
do a literature search to see if their methods are applicable to your
L. Scot Bastian Ph.D. 206-762-1010 ext. 1918 (voice)
University of Washington at the 206-764-2689 (FAX)
Veterans' Affairs Medical Center yacman at u.washington.edu (Email)
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Seattle WA 98108