Re: [ccp4bb]: Protein-DNA complex

["Shekhar C. Mande" <shekhar@www.cdfd.org.in>]

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We have been using G250 (comassie brilliant blue) to stain protein
crystals, which in principle should not stain DNA.  You could use
something like acridine orange, which in principle should not stain
proteins (I guess, perhaps I am wrong).

Shekhar

=======================================================

  Shekhar C. Mande, Ph. D.          Phone:
  Centre for DNA Fingerprinting     +91-40-27171442 (O)
         and Diagnostics            +91-40-27158535 (R)
  ECIL Road, Nacharam
  Hyderabad- 500 076 INDIA          Facsimile:
  Email: shekhar@www.cdfd.org.in    +91-40-27155610
         shekhar_mande@hotmail.com

========================================================

On Thu, 27 Mar 2003, Laurence Pearl wrote:

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>
> Tricky one ... you should also consider the possibility that the
> crystals are uncomplexed DNA - duplexes in the 10-13 base-pair range
> crystallise depressingly easily (not depressing if you are a DNA
> crystallographer of course). IZIT dye doesn't help here as DNA-also
> crystals are also stained. Do  unit-cell volume calculations give no
> clues as to probable a.u. content ?
>
> If crystals are big enough/plentiful you could fish some out, wash
> thoroughly and run them into a non-denaturing gel, and stain it with
> ethidium bromide - this binds dsDNA. You can also stain the same gel
> with Coomassie to visualise the protein. Alternatively try and image the
> gel from the fluoresence of your built-in fluorophore. If you have lots
> of crystals and/or they are really big, you could look at the UV
> spectrum of the dissolved crystals and measure the 260nm/280nm ratio.
>
> Have fun ....
>
>
> Elias J. Fernandez wrote:
>
> >***  For details on how to be removed from this list visit the  ***
> >***          CCP4 home page http://www.ccp4.ac.uk         ***
> >
> >Folks,
> >
> >Is there a clever way to tell if our crystals are complexed protein:DNA
> >versus unbound protein, without having to wait until we have maps? We have a
> >fluorophore attached to the DNA fragment.
> >
> >
> >Elias J. Fernandez, PhD
> >Assistant Professor
> >University of Tennessee
> >Biochemistry, Cellular and Molecular Biology
> >Knoxville, TN 37996-0840
> >Tel. No. 865-974-4090
> >Fax. No. 865-974-6306
> >e-mail: elias.fernandez@utk.edu
> >
> >
> >
> >
> >
>
> --
>
> ---------------------------------------------------------------------------
>   Laurence H. Pearl
>
>   Section of Structural Biology, Institute of Cancer Research
>   Chester Beatty Laboratories,  237 Fulham Road
>   London SW3 6JB, UK
>
>   Phone +44-207-970 6045 : Secretary +44-207-970 6046
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