Re: [ccp4bb]: Protein-DNA complex

[Philippe BENAS <benas@chemistry.montana.edu>]

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Hi Folks,

There is a problem trying to evaluate the presence of nucleic acids by
gel experiments.
If for some reasons the crystals are not well washed enough, you could
see a band corresponding to your DNA, but bound to the surface of the
crystals (I have a very good example of that on HIV-1 RT/RNA
complexes...).
So, to solve this problem you will have to quantify your bands and the
volume your crystals (same problem with an end labelling of your NA). At
this point, it seems easier to me to take a UV absorbance spectrum and
use the absorbance profile of the protein alone and the DNA alone to
deconvolute your experimental profile. I also have a very good example
of that on the same subject. In addition, you also get the stoechiometry
of the complex and a crude estimate of the number of molecules per au.
The only caveats are to have i) crystals of rather simple shape and ii)
a camera that is resolutive enough to get a precise estimate of the
volume (and have a 5 uL UV cuvette).

Philippe
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Philippe BENAS

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Department of Chemistry and Biochemistry
Biological X-ray Crystallography
108, Gaines Hall - Montana State University
Bozeman, MT 59717, USA

Email: benas@chemistry.montana.edu
Phone: +1 406-994-3654                       Fax: +1 406-994-5407 
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