Finding Target Genes for DNA Binding Proteins

L. Bastian yacman at homer18
Fri Oct 4 08:04:21 EST 1996


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
problem.

Good Luck.

--
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)
M/S 151 
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Seattle WA 98108 




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