Questions about DNase I FootPrinting

[Frank O. Fackelmayer]

XW wrote:

> Hi, folks,
>
> I've been using the DNase FootPrinting as an approach
> to sorting out the transcriptional factor binding
> sites within the promoter of a human
> inflammation-related gene. I've initiated some pilot
> experiments to optimize the experimental conditions,
> and the following listed some of the problems I have
> encountered. As the autograph shows that the top bands
> are much more intensive than the lower bands, I came
> up with a thought that the DNaseI digestion might be
> insufficient so that the most DNA probe is not cut at
> all by the enzyme. I increased the use of DNaseI but
> the problem remains. Then I came to realize that the
> binding rxn solution contains a high amount of salt
> since I use the crude neclear extracts (containing
> ~420 mM of NaCl), and the DNase I activity is normally
> reduced at salt concentration above 100 mM. So here
> comes my question that by what possible approach(es) I
> could lower the salt concentration in the nuclear
> extract while keeping the extract proteins active
> simultaneously. Any suggestions will be hightly
> appreciated.
>

I guess your protein won´t bind to DNA at 420mM NaCl, so dilution will
be a must. The easiest way is to simply dilute the extract fourfold with
water, then spin for 15min at full speed in a microfuge. Use the
supernatant for binding assay (some proteins will precipitate upon
dilution).
In case your protein precipitates upon dilution with water, you can try
different buffers that may enhance solubility. One possibility would be
fourfold dilution with 10mM Tris-HCl, pH 8.0, 0.4% NP-40.  Of course
your protein may have special requirements for DNA binding (DTT, ATP,
Mg2+), and there may be quite some work ahead to figure out...

Frank