How to detect GFP via FACS?

Laurentiu COCEA cocea at citi2.fr
Mon Aug 7 06:59:23 EST 1995


said at sun1.tumorbio.uni-freiburg.de (Said Hashemolhossein) wrote:
>
> Hi,
> 
>   I try to detect GFP after transfection in NIH3T3 or 293 without
> success.  Transfection is for sure not the problem ( I am able to
> transfect with luciferase + detect in luminometer ). 
>   I want to detect the
> GFP containing cells via FACS (Becton Dickinson , FL1H / FL2A) , but
> cannot see anything.
>   Even under the microscope it is not possible to
> detect positive cells. 
> 
> 
> Does anyone can help???....
> 
> 
> thanks a lot ....   
> 
> 
> said
> 
> 
> -------------------------------------------------------------------------------
> 
> Dr. Said Hashemolhosseini	phone (761)2061531
> Tumor Biology Center		fax   (761)2061505
> Dep Exp Cancer Research		email said at sun1.tumorbio.uni-freiburg.de
> Breisacherstr. 117
> D-79106 Freiburg, Germany
> -------------------------------------------------------------------------------
> 
> 
Hi, Said,

Here's a copy of a message I got from Clontech some time ago:

>For FACS, GFP expression can be detected under the 
>following wavelength: excitation @488 nm and emmission @525 nm. 

No, there seems to be one more problem: someone said (not Simon!)
that the GFP currently available is not well expressed in some
cells because it is a gelly-fish protein and this beast has a 
different codon usage than vertebrates; this information has been 
posted to this newsgroup a few months ago but I don't remember 
by whom. Apparently, someone has worked out a solution by mutating
the original DNA sequence in order to have a different codon usage
and managed to express the protein in mammal cells. But who, who?
If you find out who can provide this modified gene I would greatly
appreciate if you let me know.

Regards,

Laurentiu





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