eGFP/mCherry FRET

[lechristophe]

Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal I would like to perform simple FRET measurements (with a three-filter method) to detect interaction between two proteins in living cells. Most of the people use a CFP/YFP couple (or variants like mCerrulean/mCitrine), but I would prefer to use an eGFP/mCherry couple. Do you know if this pair would be good for FRET ? There was a paper showing that it could be the case ( http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=16941642 ) but there was also a discussion on this list saying that mCherry behaves poorly as a FRET acceptor, due to its amino and carboxy  terminus sequences  ( http://listserv.buffalo.edu/cgi-bin/wa?A2=ind0707&L=confocal&D=0&P=16580).

So what is your advice, should I try to see eGFP/mCherry FRET or directly order a three-filer CFP/YFP set and start to switch the FPs on my constructs ? Another question : do you have a good positive control for FRET that works well in hippocampal neuronal cultures ?

Thank you for your advices,


Christophe Leterrier

Postdoc
INSERM UMR641 Neurobiology of ionic channels
IFR Jean Roche - Mediterranee University
Marseille, France

[Joachim Goedhart]

Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

Dear Christophe,

We recently published a paper (PMID: 17925859) on red FRET acceptors:
http://dx.doi.org/10.1371/journal.pone.0001011
We concluded that YFP is preffered as donor over GFP when mCherry is the acceptor, based on R0
values (the YFP-mCherry construct is characterized in the above-mentioned paper and is available
as a positive control)
In your filter-cube approach, you are essentially measuring sensitized emission. So you would
benefit from an acceptor with a high quantum yield. (This is why CFP-YFP is such a nice FRET pair).
If you want to stick to GFP as a donor, you're probably better of using tagRFP or mStrawberry as an
acceptor.

Regards,
Joachim.

Dr. Ir. Joachim Goedhart
Section Molecular Cytology
Swammerdam Institute for Life Sciences
University of Amsterdam
Kruislaan 316
NL-1098 SM Amsterdam
The Netherlands
Tel: [phone]
Fax: [phone]
http://wwwmc.bio.uva.nl/~joachim

[F Javier Díez Guerra]

Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

Christophe,

Adding to Joachim comments, you should also
consider that red-shifted donor-acceptor pairs
show wider tails of excitation and emission.
Thus, if your are thinking on sensitized emission
to measure FRET, be aware that cross-excitation
(acceptor) and bleed-through (donor) values are higher than those of CFP-YFP.

For a discussion have a look at
Fluorescent protein FRET: the good, the bad and
the ugly.(2007) Piston, DW and Kremers, GJ. Trends Biochem Sci. 32(9):407-14.

Regards,


At 09:22 25/01/2008, you wrote:

>Search the CONFOCAL archive at
>http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
>Dear Christophe,
>
>We recently published a paper (PMID: 17925859) on red FRET acceptors:
>http://dx.doi.org/10.1371/journal.pone.0001011
>We concluded that YFP is preffered as donor over
>GFP when mCherry is the acceptor, based on R0
>values (the YFP-mCherry construct is
>characterized in the above-mentioned paper and is available
>as a positive control)
>In your filter-cube approach, you are
>essentially measuring sensitized emission. So you would
>benefit from an acceptor with a high quantum
>yield. (This is why CFP-YFP is such a nice FRET pair).
>If you want to stick to GFP as a donor, you're
>probably better of using tagRFP or mStrawberry as an
>acceptor.
>
>Regards,
>Joachim.
>
>Dr. Ir. Joachim Goedhart
>Section Molecular Cytology
>Swammerdam Institute for Life Sciences
>University of Amsterdam
>Kruislaan 316
>NL-1098 SM Amsterdam
>The Netherlands
>Tel: [phone]
>Fax: [phone]
>http://wwwmc.bio.uva.nl/~joachim

F Javier Diez-Guerra, PhD
Profesor Titular
Centro de Biologia Molecular Severo Ochoa
C/ Nicolás Cabrera, 1
Universidad Autónoma
Ctra Colmenar Viejo Km 15
Cantoblanco, 28049 Madrid
SPAIN

phone:  [phone]
e-mail: [hidden email]

[lechristophe]

Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Thank you for these usefull advices and references. Do anyone know where (web, article...) I can find primers and hints on how to design fusion proteins to optimize the chance to observe FRET ?

Christophe Leterrier

On Jan 25, 2008 10:05 AM, F Javier Diez Guerra <[hidden email]> wrote:

Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

Christophe,

Adding to Joachim comments, you should also
consider that red-shifted donor-acceptor pairs
show wider tails of excitation and emission.
Thus, if your are thinking on sensitized emission
to measure FRET, be aware that cross-excitation
(acceptor) and bleed-through (donor) values are higher than those of CFP-YFP.

For a discussion have a look at
Fluorescent protein FRET: the good, the bad and
the ugly.(2007) Piston, DW and Kremers, GJ. Trends Biochem Sci. 32(9):407-14.

Regards,

At 09:22 25/01/2008, you wrote:
>Search the CONFOCAL archive at
>http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
>Dear Christophe,
>
>We recently published a paper (PMID: 17925859) on red FRET acceptors:
>http://dx.doi.org/10.1371/journal.pone.0001011
>We concluded that YFP is preffered as donor over
>GFP when mCherry is the acceptor, based on R0
>values (the YFP-mCherry construct is
>characterized in the above-mentioned paper and is available
>as a positive control)
>In your filter-cube approach, you are
>essentially measuring sensitized emission. So you would
>benefit from an acceptor with a high quantum
>yield. (This is why CFP-YFP is such a nice FRET pair).
>If you want to stick to GFP as a donor, you're
>probably better of using tagRFP or mStrawberry as an
>acceptor.
>
>Regards,
>Joachim.
>
>Dr. Ir. Joachim Goedhart
>Section Molecular Cytology
>Swammerdam Institute for Life Sciences
>University of Amsterdam
>Kruislaan 316
>NL-1098 SM Amsterdam
>The Netherlands
>Tel: [phone]
>Fax: [phone]
>http://wwwmc.bio.uva.nl/~joachim

F Javier Diez-Guerra, PhD
Profesor Titular
Centro de Biologia Molecular Severo Ochoa
C/ Nicolás Cabrera, 1
Universidad Autónoma
Ctra Colmenar Viejo Km 15
Cantoblanco, 28049 Madrid
SPAIN

phone:  [phone]
e-mail: [hidden email]