DNase FOOTPRINTING Off a Gel-Retarded Complex

Ken Doyle kdoyle at promega.com
Tue Dec 6 16:01:14 EST 1994


In article <1994Dec2.145747.17546 at tin.monsanto.com>, Sandy Curtiss <swcurt at ccmail.monsanto.com> says:
>
>In article <3bde8u$l6p at bigfoot.wustl.edu> Thiruvamoor P. Ramkumar,
>ramu at artsci.wustl.edu writes:
>>Hi,
>>       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. 
>>       
>>
>> _
>>ramu
>
>A protocol for doing DNase footprinting directly in a mobility shift gel
>is given in Kuwabara, MD and Sigman, DS (1987) Biochemistry 26:
>7234-7238.  I have not used this protocol, but it was used successfully
>by Wolfe et al. (1993) JBC 268: 12418-12426.  Hope this helps.
>
>Sandy Curtiss
>Monsanto Co.
>swcurt at ccmail.monsanto.com


The Kuwabara and Sigman reference actually describes the use of
copper-phenanthroline, not DNAse I, as the footprinting agent.
However, the same technique can be used with standard DNAse I reaction
conditions (for example, Cockerell et al, (1989) Mol. Cell. Biol.,
9: 2464-2476), with the caveat that you need about 10x more DNAse I 
as you would for solution footprinting.  The exact amount depends 
on the volume of the gel slice (I typically used about 50 ug/ml DNAse I
and reaction times of 1-3 min.)

Ken
kdoyle at promega.com



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