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faster fluorescence

'Mike' Michael J. Moser moser at U.WASHINGTON.EDU
Wed May 15 18:04:38 EST 1996


Peter,
	I noticed on their Web page that Clonetech (a sponsor of this
newsgroup) now offers humanized versions of a "super" GFP that was
isolated using FACS by Brendan Cormack and Raphael Valdivia in Stanley
Falkow's lab at Stanford.  The mutations contained in that mutant are
F64L, S65T.  You can find more information on the WWW at:

http://www.clontech.com/clontech/APR96UPD/EGFP.html

	Brendan also isolated 2 other mutants.  One contains the following
mutations S65A, V68L and S72A.  It is supposed to be even brighter than
F64L, S65T.  Another mutant isolated was S65G, S72A.  All three mutants
are to be published in the July? 1996 issue of Gene.

	We found that mutant 2 was extremely bright whenm expressed in
S. cerevisiae.  Hope that this info is of some use.

Happy GFPing!

Mike Moser                                            Tel: 206-543-6585
UW Department of Pathology                            FAX: 206-543-3644
Box 357470                                       moser at u.washington.edu
Seattle, WA  98195                 http://weber.u.washington.edu/~moser

On 13 May 1996, Lotti wrote:

> I'm using GFP as a quantitative marker to on-line and flow cytometrically
> track protein expression in Saccharomyces cerevisiae. Since, due to its
> folding and autooxidation, at least 2.5 h (wt GFP in centromeric plasmid)
> and even up to 4.5 h (wt GFP in 100-copy plasmid) are needed to obtain final
> fluorescence, wt GFP turned out to be useless in this respect. Also GFPS65T
> remains suboptimal since, when even cloned in a centromeric plasmid, still
> 45 min to 1 h are needed. Fixing the cells and vortexing them to stimulate
> autooxdation does not help. In fact, the fluorescence most of the time
> completely disappears probably due to a stimulated entering of the fixing
> agent inside the cells (e.g also when using EtOH70%, contrary to many
> literature reports,...). Besides we'd like to track GFP expression without
> disturbing the cells (e.g. by fixation). Are there some new and faster GFPs
> around of which I do not know ? Any suggestions on how to stimulate the
> folding/autoxidation in vivo without disturbing the cells ?
>
> Many thanks,
>
>
> Peter De wulf
>
>
>





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