[Date Prev][Date Next][Thread Prev][Thread Next][Date Index][Thread Index]
Re: [ccp4bb]: Protein-DNA complex
*** For details on how to be removed from this list visit the ***
*** CCP4 home page http://www.ccp4.ac.uk ***
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
FAX +44-207-970 6051 : E-Mail l.pearl@icr.ac.uk
---------------------------------------------------------------------------
" Live Simply and do Serious Things .. " - Dorothy Crowfoot Hodgkin
---------------------------------------------------------------------------