GFP on SDS-GEL

Rich Dudley rdudley+ at pitt.edu
Wed Sep 23 09:20:17 EST 1998


Jan-Peter Siebrasse wrote:

> Rich Dudley wrote:
> >
> > On SDS-PAGE, no.  I'm not sure about native PAGE, but probably.
> >
> > rich
> >
> From my own experience, yes. Most of the GFP/GFP fusion protein will not
> be denatured as long the sample will not be heated or your gel is
> running too hot. And you have to load your sample immediatly after
> mixing them with sample buffer. After fixation fluorescence will be
> lost, of course. I even noticed a clearly visible fluorescent band after
> transferring GFP/GFP fusionproteins onto a NC membrane for Western
> Blots.
>
> BTW, the non-denatured GFP runs at an apparently lower molecular weight
> (ca. 8kDa "smaller" than the denatured protein). Most likely due to the
> more compact structure of the native protein I think.
>
> Jan-Peter Siebrasse

 Just out of curiosity, how do you visualize the green--with a
transilluminator?

I usually include boiling in SDS-PAGE buffer as part of my protocol.  I'll
have to try this!

rich

--
--- --- --- -- -- -- --- --- ---
Richard J. Dudley (rdudley+ at pitt.edu)
Research Specialist V
Dept. of Cell Biology and Physiology
University of Pittsburgh
http://www.cbp.pitt.edu
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