Isol. of Transcription factors

Sean Stevens stevens at rockvax.rockefeller.edu
Mon Jul 3 23:14:41 EST 1995


schraute at uni-bonn.de (Bernhard Schrautemeier) wrote:
>I am looking for a method for positive selection of (cyano)bacterial
>transcription factors expressed in (E. coli) gene libraries to isolate the
>corresponding gene.  
>
>Also I heard that it is possible to bind promoter-containing DNA sequences
>to certain matrices (e. g. latex; J. Biomater. Sci. Polym. Ed. 5(4),
>293-302, 1994; I don´t have access to this journal) and thus to
>selectively purify binding proteins, e.g. for use in footprinting.
>
>I would be glad to get some help!
>
>-- 
>____________________________________________________________
>Bernhard Schrautemeier      Tel: +49-228-73-5277 or 5536
>Botanisches Institut            Fax: +49-228-73-5513
>der Universitaet Bonn        E-mail: schraute at uni-bonn.de
>Kirschallee 1              x________________________________
>53115 Bonn                 x
>Germany                    x
>___________________________x

Bernhard-
 
There is a much easier method that should work for you. It uses matrix
attachment, but reverses it to be the bacterial proteins bound to the
nitrocellulose and then you probe this with the radiolabelled DNA of 
interest. Thus you would express the library proteins, stick them to 
the filters, denature, renature, and expose to DNA, wash and use the 
spots on the blots to pick the bacteria bearing the desired gene. A few
rounds of purification and you should have it. The technique is referred
to as -southwestern screening- and an explanation and protocol is found
in 'Current Protocols' or 'The Red Book' as it is sometimes called. I 
can supply you with references besides.

To biochemically isolate factors I would strongly suggest columns work-
you may have to fractionate initially using phosphocellulose or a 
similar matrix, then use a DNA affinity method ie oligos covalently 
linked to agarose. Descriptions can be found in papers out of the 
Roeder or Tjian labs. I can also look them up for you if you so desire.
Many standard methods books such as the 'Practical Approach' series 
also contain these methods in detail.

Good luck and e-mail me with any more comments or queries.

Sean Stevens  The Rockefeller University
stevens at rockvax.rockefeller.edu





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