Fret pairs

[Scott Howell-3]

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List Members,


Have a member lab that wants to do some FRET work. They are starting
at the start, preparing to build their clones. Therefore the question
is: What at the moment seems to be the best fret pair that will give
us the best chance for success? Have worked with CFP/YFP in the past
but guessing there is something superior these days. Thanks.

Scott J. Howell, Ph.D.
Manager, Imaging Module
Visual Sciences Research Center
Case Western Reserve University
2085 Adelbert Rd.
Institute of Pathology Room 106
Cleveland, Ohio 44106
216-368-2300
http://www.case.edu/med/vsrc/

[Simon Ameer-Beg]

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Dear Scott,

I think the answer would reflect which measurement method is being employed.

 From my perspective (coming from a FLIM background), I would chose
GFP-mRFP1 as a pair of choice. GFP-mCherry also works well but from
our point of view (with many constructs already in the lab) perhaps
not worth re-cloning yet.

There are certainly better versions of the CFP/YFP (which performs
badly for FLIM in our hands) which might be better suited for
ratiometric FRET, given the pitiful QY of mRFP.

Best wishes

Simon

At 18:56 18/03/2008, you wrote:

>Search the CONFOCAL archive at
>http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
>List Members,
>
>
>Have a member lab that wants to do some FRET work. They are starting
>at the start, preparing to build their clones. Therefore the question
>is: What at the moment seems to be the best fret pair that will give
>us the best chance for success? Have worked with CFP/YFP in the past
>but guessing there is something superior these days. Thanks.
>
>Scott J. Howell, Ph.D.
>Manager, Imaging Module
>Visual Sciences Research Center
>Case Western Reserve University
>2085 Adelbert Rd.
>Institute of Pathology Room 106
>Cleveland, Ohio 44106
>216-368-2300
>http://www.case.edu/med/vsrc/

Dr Simon M. Ameer-Beg

Non-clinical lecturer
The Richard Dimbleby Department of Cancer Research
Randall Division of Cell & Molecular Biophysics and Division of Cancer Studies
King's College London
2rd Floor, Room 2.30A
New Hunt's House, Guy's Medical School Campus
London SE1 1UL

[Sam's Mail]

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Simon,

In your lab using FLIM, have you compared GFP-mCherry and GFP-mRFP1?  You mentioned that they both work...but which works better in your hands?  How about comparing either of these in a FLIM scenario to the common set up of Cerulean-Venus that many labs employ.

Thanks for the insight.

Cheers,

--
Samuel A. Connell
Director of Light Microscopy
Cell & Tissue Imaging Center
St. Jude Children's Research Hospital
332 North Lauderdale St., E7061
Memphis, TN 38105-2794
Office [phone]
Cell [phone]
[hidden email]


-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Simon Ameer-Beg
Sent: Wednesday, March 19, 2008 6:29 AM
To: [hidden email]
Subject: Re: Fret pairs. .

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http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

Dear Scott,

I think the answer would reflect which measurement method is being employed.

 From my perspective (coming from a FLIM background), I would chose
GFP-mRFP1 as a pair of choice. GFP-mCherry also works well but from
our point of view (with many constructs already in the lab) perhaps
not worth re-cloning yet.

There are certainly better versions of the CFP/YFP (which performs
badly for FLIM in our hands) which might be better suited for
ratiometric FRET, given the pitiful QY of mRFP.

Best wishes

Simon

At 18:56 18/03/2008, you wrote:

>Search the CONFOCAL archive at
>http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
>List Members,
>
>
>Have a member lab that wants to do some FRET work. They are starting
>at the start, preparing to build their clones. Therefore the question
>is: What at the moment seems to be the best fret pair that will give
>us the best chance for success? Have worked with CFP/YFP in the past
>but guessing there is something superior these days. Thanks.
>
>Scott J. Howell, Ph.D.
>Manager, Imaging Module
>Visual Sciences Research Center
>Case Western Reserve University
>2085 Adelbert Rd.
>Institute of Pathology Room 106
>Cleveland, Ohio 44106
>216-368-2300
>http://www.case.edu/med/vsrc/

Dr Simon M. Ameer-Beg

Non-clinical lecturer
The Richard Dimbleby Department of Cancer Research
Randall Division of Cell & Molecular Biophysics and Division of Cancer Studies
King's College London
2rd Floor, Room 2.30A
New Hunt's House, Guy's Medical School Campus
London SE1 1UL

[Joachim Goedhart]

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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: [phone]
Fax: [phone]
http://wwwmc.bio.uva.nl/~joachim

[Karl Garsha]

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Hello Scott,

To some degree the answer to your question depends on the type of FRET
detection strategy.

Those GFP variants that are the most efficient donor-acceptor pairs for
FRET measurements show a high degree of spectral overlap; this tends to
confound accurate analysis by conventional filter-based imaging systems.
  Spectral imaging and un-mixing technology provides the ability to
succesfully use FRET pairs with greater overlap, such as GFP-YFP
(Zimmermann et al., 2002).  The study demonstrates that linear un-mixing
of convolved spectral signatures for FRET analysis can be performed
reliably with relatively few, in this case four, wavelength channels
contributing to the dataset.

By developing a spectral unmixing algorithm (sRET analysis) that
accounts for the effects of resonant energy transfer on the intensity
distribution of spectral components eminating from mixtures of donor and
acceptor molecules, Thaler et al. showed that spectral imaging and
subsequent computational post-processing can be used to accurately
measure donor and acceptor concentrations as well as their FRET
efficiencies.

Sophisticated quantitative FRET methods reported by Gu et al. and Raicu
et al. both involve combining spectral imaging and acceptor photobleaching.

Gu, Y, Di, WL, Kelsell, DP, and Zicha, D. (2004). ‘Quantitative
fluorescence resonance energy transfer (FRET) measurement with acceptor
photobleaching and spectral unmixing.’ J. Microsc. 215, 2: 162-173.

Raicu, V, Jansma, DB, Dwayne Miller, RJ, and Friesen, JD. (2005).
‘Protein interaction quantified in vivo by spectrally resolved
fluorescence resonance energy transfer.’ Biochem. J. 385: 265-277.

Thaler, C, Koushik, SV, Blank, PS, Vogel, SS. (2005). 'Quantitative
Multiphoton Spectral Imaging and its use for Measuring Resonance Energy
Transfer.' Biophys. J. 89: 2736-2749.

Zimmerman, T, Rietdorf, J, Girod, A, Georget, V, Pepperkok, R. (2002).
'Spectral imaging and linear un-mixing enables improved FRET efficiency
with a novel GFP2-YFP FRET pair.' FEBS Letters 531: 245-249.

Cheers,
Karl


Scott Howell wrote:

> Search the CONFOCAL archive at
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
> List Members,
>
>
> Have a member lab that wants to do some FRET work. They are starting
> at the start, preparing to build their clones. Therefore the question
> is: What at the moment seems to be the best fret pair that will give
> us the best chance for success? Have worked with CFP/YFP in the past
> but guessing there is something superior these days. Thanks.
>
> Scott J. Howell, Ph.D.
> Manager, Imaging Module
> Visual Sciences Research Center
> Case Western Reserve University
> 2085 Adelbert Rd.
> Institute of Pathology Room 106
> Cleveland, Ohio 44106
> 216-368-2300
> http://www.case.edu/med/vsrc/

--
Karl Garsha
Research Microscopy Specialist
US-Southwest Region
Leica Microsystems-Life Sciences Research
www.leica-microsystems.com