FRET with 3 fluorophores

[Peter Egri]

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Hello,

We would like to perform FRET experiments to detect the changes in interaction between three
proteins using three fluorophores in living cells.
We tried to use CFP-YFP-mCherry system but we did not find FRET between neither the known
interacting proteins nor the fused YFP-mCherry. (CFP-YFP FRET was OK).

Is the problem with the Cherry? I read here in a previous discussion that the mRFP could be
better than the Cherry so we should use a different red FP?

Tanks,
Peter

[yuansheng sun]

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Dear Peter,

We have used the following combinations for our 3-color FRET work and
obtained positive results -

CFP-Venus-mCherry
Teal-Venus-mRFP
Teal-Venus-tdTomato

For the Teal-Venus-tdTomato combination, please see -
Y. Sun, H. Wallrabe, C.F. Booker, R.N. Day and A. Periasamy. “Three-color
spectral FRET microscopy localizes three interacting proteins in living
cells”, *Biophysical Journal* 99, 1274-1283 (2010)

The concern about mCherry (if you detect FRET from sensitized mCherry on
a confocal) is that it is a dark red probe and a regular PMT usually has a
poor quantum efficiency on red wavelengths.  In this concern, mRFP is a
better choice.  Have you done your fused YFP-mCherry FRET on a fluorescence
lifetime imaging system?  There, you measure the YFP fluorescence lifetime
and do not need to worry about the mCherry fluorescence - it is just a
quencher.

Best regards,
Sheng




2011/10/6 Peter Egri <[hidden email]>

> *****
> To join, leave or search the confocal microscopy listserv, go to:
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> *****
>
> Hello,
>
> We would like to perform FRET experiments to detect the changes in
> interaction between three
> proteins using three fluorophores in living cells.
> We tried to use CFP-YFP-mCherry system but we did not find FRET between
> neither the known
> interacting proteins nor the fused YFP-mCherry. (CFP-YFP FRET was OK).
>
> Is the problem with the Cherry? I read here in a previous discussion that
> the mRFP could be
> better than the Cherry so we should use a different red FP?
>
> Tanks,
> Peter
>

[Peter Egri]

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Dear Sheng,

Thank you for your advices. We planned both mRFP and mCherry with EYFP to
see the performance of these system in our hands but unfortunately there was
some problem with the cloning of RFP constructs so we started with the mCherry
only.

At this time our microscopy system is not usable for FLIM detection of FRET so
we are restricted to the intensity-based method.

So the mRFP should be better?
Another idea: BFP-GFP-mOrange could work in 3-FRET system?

Sincerely,
Peter

[yuansheng sun]

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Dear Peter,

For myself, I have never used BFP.  But I know BFP-GFP should work for FRET.
 Dr. Ammasi Periasamy, director of the Keck center where I am
working, used BFP-GFP for FRET a long time ago.  I can send you a couple of
references if you want.  In my opinion, I would like to avoid BFP for
live-cell imaging unless I have no other choice, because BFP requires UV
excitation.  If you use 2P, the bleedthrough corrections will be much more
complicated.

The YFP-mCherry fusion should give you some FRET.  You did not see any FRET
signal at all?  Or you saw a lower FRET efficiency for YFP-mCherry
 compared another fusion construct e.g. CFP-YFP?

Best regards,
Sheng

Yuansheng Sun, Ph.D.
Research Scientist
W.M. Keck Center for Cellular Imaging: www.kcci.virginia.edu
University of Virginia
FRET Workshop: www.kcci.virginia.edu/workshop/workshop2012


2011/10/10 Peter Egri <[hidden email]>

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Dear Sheng,
>
> Thank you for your advices. We planned both mRFP and mCherry with EYFP to
> see the performance of these system in our hands but unfortunately there
> was
> some problem with the cloning of RFP constructs so we started with the
> mCherry
> only.
>
> At this time our microscopy system is not usable for FLIM detection of FRET
> so
> we are restricted to the intensity-based method.
>
> So the mRFP should be better?
> Another idea: BFP-GFP-mOrange could work in 3-FRET system?
>
> Sincerely,
> Peter
>

[Peter Egri]

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Dear Sheng,

I did not expect similar efficiency compare to the CFP-YFP, but the average of
YFP-mCherry was only ~4% and in case of our interested proteins it was near
zero (in CFP-YFP system they work fine). Next step will be the testing of mRFP I
hope it will work better.

The BFP was an idea and beside its disadvantages as you mentioned it would
mean a lot of subcloning and optimizing. Even so I would appreciate if you could
send some reference about the application of the BFP-based method.

Best regards,
Peter

[yuansheng sun]

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Dear Peter,

I will send you the BFP-GFP references directly (off the list) soon.  We can
also discuss further off the list on this topic if you want.

Sheng

2011/10/11 Peter Egri <[hidden email]>

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Dear Sheng,
>
> I did not expect similar efficiency compare to the CFP-YFP, but the average
> of
> YFP-mCherry was only ~4% and in case of our interested proteins it was near
> zero (in CFP-YFP system they work fine). Next step will be the testing of
> mRFP I
> hope it will work better.
>
> The BFP was an idea and beside its disadvantages as you mentioned it would
> mean a lot of subcloning and optimizing. Even so I would appreciate if you
> could
> send some reference about the application of the BFP-based method.
>
> Best regards,
> Peter
>