Sv: re DNA binding site

Jens Tornoe jens at tornoe.net
Fri Jun 9 14:51:53 EST 2000


If you have no idea whatsoever about to which sequence/promoter your protein
binds to, I guess some kind of screening would need to be done. This might
be a far out experiment to suggest, but some kind of gel-mobility shift
assay with randomized short dsDNA oligos could work. This might be done as
follows:

- Order an oligo with the sequence R1-NN...NN-R2. (R1 and R2 are suitable
restriction sites).
- Make it double stranded with a complementary primer covering the 3'
restriction site.
- Digest with the two restriction enzymes.
- Label the DNA (better make the R1 and R2 have 5' overhangs, then).
- Make a gel-shift assay with your purified protein and your labeled oligos
with LOTS of oligo.
- Excise retarded protein/DNA complex and purify the DNA.
- Clone it in a suitable vector and sequence clones.

The big question is whether you will be able to obtain enough DNA for the
cloning step. An alternative to radioactively labeling the DNA might be able
to His-tag your protein, incubate it with Ni-NTA + randomized oligo and
elute the DNA.

Hope you find this somewhat useful.

Jens

--
Jens Tornøe
NsGene
Pederstrup, Denmark

"DR JULIAN R MARCHESI" <Marchesi at Cardiff.ac.uk> skrev i en
nyhedsmeddelelse:E1303WT-00037P-00 at dove2.cf.ac.uk...
> I think I have identified a DNA binding protein ( a putative
> transcriptional regulator).
> what would be the best method for identifiying the site to which it
> binds.  I have no
> idea where it binds, but a quick method to find the piece of DNA
> would be great.
>
> cheers
>
> Julian
>
>
>
> **************************************
> Dr Julian R Marchesi
> Cardiff University
> School of Biosciences
> (Biology)
> PO Box 915
> Cardiff
> CF10 3TL
> United Kingdom
>
> Tel: (+44) 029 20875073
> Fax: (+44) 029 20874305
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