Green Fluo. Prot.

Mic Chaudoir mic at nwu.edu
Fri Jan 20 01:26:16 EST 1995


Hi there,

we have been playing with transforming ES cells (R1) with GFP under
control of the cmv promoter that others have used.  Initially, experiments
were done using co-transformation with the GFP plasmid, and a neo
plasmid.  These experiments were mostly failures, but we did see some
colonies which appeared to be expressing.  Expression was detected by
confolcal microscopy for flourscein, with excitation at 455nm (I think)
and emission at around 510nm  (i.e. filter for 500-515 nm, I think).  We
did notice that we had a really high background on glass slides.  We are
currently examining a new batch of cells transformed with a cmv-GFP and
neo on the same plasmid.  No results yet.  

I too have heard that not all cells can carry out the cyclization reaction
to activate the GFP flourophore.  However, the initial GFP papers seems to
indicate that the cyclization was self-catalyzed by the protein.  This
makes sense to me, since GFP works in bacteria!!!  (You would imagine that
the bacteria don't wouldn't have co-factors in common with a
jellyfish.....).  Maybe the reason that GFP doesn't work in some cells is
due to another reason ?  Lastly, I have heard of a Red Flourscent Protein
(RFP), also from one of our tentacled-freinds.  Does anyone know how this
protein stacks up to GFP ??

-- 
mic
mic at nwu.edu
 
"It [PowerPC Mac] won't have any effect at all on Intel machines.
They will continue to plod along, running the same clunky Windows              and wretched DOS drivel they always have.  The PPC will affect Intel PCs the same way an SR-71 Blackbird affects a dairy cow."
                     Robert Rhode, in comp.sys.mac.advocacy



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