GFP in HeLa cells

David Micklem drm21 at mole.bio.cam.ac.uk
Mon Apr 28 08:50:56 EST 1997


In article <199704280700.JAA15216 at fmi.ch>, ludin at FMI.CH (Beat Ludin) wrote:

>Joe Binder wrote:
>
>>I have put GFP (ser65thr and ile167thr) into Mengo virus and am using 
>>fluorescent microscopy to visualize infected cells.  I have seen low 
>>expression levels.  Any advice on preparing/visualizing slides to see 
>>the protein better?
>
>1. Using a low-riboflavin medium or a blanaced salt solution should lower 
>the background. 
>2. Don't fix the cells to get the highest fluorescence output.
>3. If fixation is needed, paraformaldehye seems to be the least damaging. 
>Readjust the pH to 6.5 or higher.
>
>BTW, I'm not aware of a S65T/I167T mutant, what is its spectrum? Can you 
>give me a reference?
>
>Beat
>
Hi Beat,

You might look at:

1. Heim R, Prasher DC, Tsien RY. Wavelength mutations and posttranslational
auto oxidation of green fluorescent protein. Proc. Natl. Acad. Sci USA
1994, 91:12501-12504.
2. Heim R, Cubitt AB, Tsien RY. Improved green fluorescence. Nature 1995,
373:663-664.
3. Brand AH. GFP in Drosophila. Trends in Genetics 1995, 11:324-325.

The single mutants shift the excitation spectrum to a single peak near
475nm, with emission at 508nm IIRC...

David

-- 
D.R.Micklem,                                Time flies like an arrow... 
Wellcome/CRC Institute,            Fruit flies like a banana.       
Cambridge CB2 1QR, UK              
Email:drm21 at mole.bio.cam.ac.uk Junk mail very very unwelcome.



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