DNase FOOTPRINTING Off a Gel-Retarded Complex
tan at mol.biol.ethz.ch
Wed Nov 30 06:06:55 EST 1994
In article <3bde8u$l6p at bigfoot.wustl.edu>, ramu at artsci.wustl.edu
(Thiruvamoor P. Ramkumar) wrote:
> i'm looking for help on doing DNase Footprinting from a
> DNA-Protein complex isolated from a gel-retarded complex. Primarily, my
> concern is to elute the DNA-Protein complex out of the gel without
> disrupting the complex. Possible buffer compositions, elution conditions,
> quantitation parameters etc. will all help.
I haven't tried to do what you're suggesting (run bandshift gel, isolate
protein/DNA complex, treat with DNaseI, run nicked DNA on sequencing type
gel), but I have performed essentially the same experiment in a different
order (treat protein-DNA mixture with DNaseI, run bandshift gel, isolate
nicked DNA from protein/DNA complex band, run nicked DNA on sequencing
type gel). Is it really necessary for you to treat gel isolated
protein/DNA complex with DNaseI for your footprinting experiment? Since
you're only going to nick the DNA (max once per DNA if possible), the
protein/nicked DNA complex should migrate at the same position in your
bandshift gel as protein/unnicked DNA unless the DNA in the complex is
distorted in some really funny way.
If you really need to treat the purified protein/DNA complex with DNaseI,
you might try treating the band directly with DNaseI. I've never tried
this and I don't know how well you can expect DNaseI to penetrate the gel,
but it might work (has been done using other smaller chemical probes).
If you decide to treat the complex first with DNaseI and then elute just
the nicked DNA from the bandshift gel, you might find the following
Treisman, R. (1986) Cell, 46, 567-574.
Hope this helps.
Institute for Molecular Biology and Biophysics
ETH-Honggerberg (Swiss Federal Institute of Technology)
8093 Zurich, Switzerland
email: tan at mol.biol.ethz.ch
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