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
>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?
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
2. Heim R, Cubitt AB, Tsien RY. Improved green fluorescence. Nature 1995,
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...
D.R.Micklem, Time flies like an arrow...
Wellcome/CRC Institute, Fruit flies like a banana.
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