Questions about DNase I FootPrinting

XW xw2k at yahoo.com
Tue Feb 22 16:03:22 EST 2000


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.

Jason Wu 
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