bleaching GFP

Nico Dantuma nico.dantuma at mtc.ki.se
Wed Feb 25 14:32:29 EST 1998


Hi,

I try to monitor with real time kinetics an EGFP fusion protein in HeLa
cells using a fluorescence microscope with a CCD camera. To my surprise I
encounter substantial bleaching of EGFP: it is not an exception that the
fluorescence decreases with 30% in 1 minute. In a control experiment I
transfected HeLa cells with EGFP (non-fused), fixed the cells with PFA and
did the same experiment. Again a strong bleaching was observed indicating
that the decrease in fluorescence with the fusion protein has nothing to do
with instability or degradation of the fusion protein. In the literature
there are many examples of monitoring GFP fusions in time. What am I doing
differently?? Could it for example be that I am using an oil immersion
objective or a too high magnification and that this gives a higher
intensity of the excitation light? Does it make sense to repeat the
experiment with the confocal microscope (fluorescence may be too low for
this)?

Any suggestions are welcome. Thanks.

Nico Dantuma



______________________
Nico Dantuma
Microbiology and Tumorbiology Center
Karolinska Institute
Box 280
S-171 77 Stockholm
SWEDEN
Phone: +46 8 7286280; fax: +46 8 330498





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