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
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Wellcome/CRC Institute, Fruit flies like a banana.
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