Questions about DNase I FootPrinting

Anton Tutter atutter at aim.salk.edu
Sun Feb 27 20:28:19 EST 2000


in article 20000222210234.9682.qmail at web3106.mail.yahoo.com, XW at
xw2k at yahoo.com wrote on 2/22/00 1:03p:

> 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.
> 

we routinely dialyse our nuclear extracts from >500mM down to 100mM KCl.  we
know the extracts remain active because we use them in in vitro
transcription reactions.  make sure you do sequential dialysis (i.e., once
at 400mM, once at 300mM, 200mM, 100mM) or you risk crashing out a lot of
proteins.  a little precipitate, however, is quite normal and extracts
should still be fine after a brief spin to remove the precipiate.

--anton





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