UV crosslinking

mbarnhart at my-dejanews.com mbarnhart at my-dejanews.com
Fri Jul 10 09:36:36 EST 1998


In article <35a5cd7e.2726037 at news.univie.ac.at>,
  a8803349.nospam at unet.univie.ac.at (Martin Offterdinger) wrote:
> On 10 Jul 1998 03:57:40 GMT, karenmk at cyllene.uwa.edu.au (Karen
> Kroeger) wrote:
>
> >I am currently trying to determine the size of a DNA-binding protein using
> >UV-crosslinking.  I bind by nuclear extract to a BrdU incorporated 32P
> >oligo, run reaction on a EMSA, then UV irradiate the gel, cut out the
> >complex, elute then run the elution on a SDS gel.  The problem is I get
> >fuzzy bands, often many bands in one lane running as a streak or smear.
> >The complex runs low on EMSA, but gives several high MW proteins on SDS,
> >which runs on SDS with a smear. How can I get clearer bands on SDS.  Any
> >help would be appreciated.
> >Thanks.  Reply to email address please.
> >
> >Karen Kroeger
> >Dept. Biochemistry
> >University of Western Australia
> >W.A., Australia
> >Email : karenmk at cyllene.uwa.edu.au
>
> Hi Karen!
> In my experience it is always a problem putting DNA on a protein gel.
> Smearing and undistinct protein bands very often arise. The only
> solution I can imagine is getting rid of the DNA before putting the
> proteins on the gel (Dnase-treatment?).
> Martin Offterdinger
> Internal Med.I,Dept. Oncology
> University of Vienna
> Austria
> E-Mail:a8803349.nospam at unet.univie.ac.at
> (remove nospam before mailing)
>

To add my .02,

Fuzzy bands are often a  problem in UV crosslinking.  Most protocols that
I've seen call for at least partial digestion of the DNA prior to SDS-PAGE. 
The problem is that your signal can go way down in this situation (if you end
label, the signal will disappear).  You may need to try a different labeled
nucleotide in making your probe, ie labeled CTP instead of ATP.

Good luck,
Michael Barnhart
Laboratory Supervisor
Connective Tissue Physiology Lab
University of Houston
Houston, Tx

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