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March 2008

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Confocal Microscopy List <[log in to unmask]>
Date:
Thu, 20 Mar 2008 05:12:49 -0400
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Confocal Microscopy List <[log in to unmask]>
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Re: Fret pairs
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Joachim Goedhart <[log in to unmask]>
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To: Scott Howell <[log in to unmask]>
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Search the CONFOCAL archive at
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Dear Scott,

I agree with Simon; CFP/YFP would be the best option for ratiometric FRET measurements 
or for methods based on measuring sensitized emission of the acceptor (e.g. filter FRET). 
I would certainly use optimized FPs: cerulean/SCFP3A and citrine/venus/SYFP2 (all with 
A206K mutations to prevent dimerization).
For donor based methods (FLIM, acceptor photobleaching) we found that yellow/red pairs 
are a substantial improvement over CFP/YFP:
http://dx.doi.org/10.1371/journal.pone.0001011
If you like you can obtain constructs that have directly linked donor-acceptor proteins as 
positive controls for FRET:
http://wwwmc.bio.uva.nl/~joachim/Resources-DNA.html

Regards,
Joachim.


Dr. Joachim Goedhart
Section Molecular Cytology
Swammerdam Institute for Life Sciences
University of Amsterdam
Kruislaan 316
NL-1098 SM Amsterdam
The Netherlands
Tel: +31(0)20 525 7774
Fax: +31(0)20 525 6271
http://wwwmc.bio.uva.nl/~joachim

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