I am a bit lost as well here.
If CFP-YPF tagged proteins
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Hi Kees,
I am a bit lost as well here.
If CFP-YPF tagged proteins are "diluted" with untagged proteins and increased FRET signal is reported, it might also point out to a more complex situation such as self-quenching of acceptor-by-acceptor and possible oligomerization (rather than dimerization of CFP-YFP tagged molecules). I would assume that CFP and/or YFP, being dimers themselves, could also stabilize any proteins that are expected to form a dimer, especially if local concentration of CFP or YFP is ca. 100-200uM or even less!!!
Monomeric CeFP or YFP might help, only a bit I would guess. Though I did lots of experiments in that direction, I did not have enough time to analyze it comprehensively. However, I have also not seen careful comparative FRET study of different FRET pairs (e.g. CeFP or YFP with or without A206K vs CeFP (YFP) with all three A206K, L221K, F223R monomeric mutations, etc.) with good and convincing statistics and accurate quantitative analysis on a voxel-by-voxel basis (or 3x3x3 VOIs).
It would be good to know what was the imaging setup used in the FRET experiment including time post transfection. I have seen lots of strange things if images were taken later that 18 hours post transfection in HELA cells with less than 1 ug of total DNA per 35mm dish.
Thus, statement of FLIM-FRET independence of concentration is only true in theory (or with highly soluble donors and acceptors in a test tube) in an environment that is free of oligomerization or non-specific aggregation of CFP/YFP tagged proteins (CFP/YFP are hydrophobic and sticky to membranes at high concentrations). And in real experiment it could be a mixture of true dimers and artefactual oligomers. And it might be difficult to separate true dimers from oligomers withing a single 3x3x3 VOI.
I can also tell you that in BiFC experiment, when mCherry was fused to split venusYFP, very strong BiFC signal (reconstituted venusYFP) has been recorded already at 12 hours post transfection pointing out either to weakness of BiFC system or non-monomeric nature of "pseudo"???-monomeric mCherry.
Thus, "clean" quantitative imaging is still a challenging task. There are some solutions to it - one of them is here - every Research Institution should have an established PI with strong background in Fundamental Biophysics, possibly with a degree in Physics rather than Biology. Promoting broadly integrated inter disciplinary environment is a priority that politicians are not aware of as they seem to be very busy with cleaning up the global economic "mess".
On an optimistic note - with new tech developments, imaging and accurate image analysis gets better very fast.
Vitaly
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From: "Straatman, Kees R. (Dr.)" <[log in to unmask]>
To: [log in to unmask]
Sent: Thursday, November 17, 2011 7:51 AM
Subject: Re: FRET-FLIM question
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Hi Guy,
Sorry, will try to explain it a little more. In the publication they found an increased FRET efficiency when an unlabelled binding partner is added to the cells and they argued that this is caused by an increase of dimerisation of their CFP and YFP labelled proteins. As FRET-FLIM is independent of concentration I would expect that there is a structural change within the dimer what brings the donor and acceptor closer together resulting in an increase of FRET efficiency. Using a Bimolecular fluorescence complementation (BiFC) system we find less signal when we add this binding partner. So I was wondering if it is possible that less dimer is formed but the dimer that is formed has a changed configuration and gives a higher FRET-FLIM signal. But as I don't know enough about FLIM I am not sure if you can see a difference in number of molecules that result in a FRET-FLIM signal by using for example the time it takes to collect a signal.
Kees
-----Original Message-----
From: Confocal Microscopy List [mailto:[log in to unmask]] On Behalf Of Guy Cox
Sent: 17 November 2011 12:32
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Subject: Re: FRET-FLIM question
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I'm a bit lost by this. As you say, donor lifetime reduction caused by FRET is independent of concentration (so long as there is no external constraint on dimer formation), but since dimer formation is typically what brings the FRET partners into close proximity, I cannot see how you can expect to have increased FRET efficiency with less dimerization. I may have misunderstood your point - if so, please clarify. My brain is probably slowing down with age!
Guy
Optical Imaging Techniques in Cell Biology
by Guy Cox CRC Press / Taylor & Francis
http://www.guycox.com/optical.htm
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Associate Professor Guy Cox, MA, DPhil(Oxon)
Australian Centre for Microscopy & Microanalysis,
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Phone +61 2 9351 3176 Fax +61 2 9351 7682
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-----Original Message-----
From: Confocal Microscopy List [mailto:[log in to unmask]] On Behalf Of Straatman, Kees R. (Dr.)
Sent: Thursday, 17 November 2011 9:34 PM
To: [log in to unmask]
Subject: FRET-FLIM question
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Dear list members,
We have a discussion about FRET-FLIM at the moment and somebody has published that they see an increase in FRET efficiency and argues that this is because there is more dimerisation between the two proteins they are interested in. Now, I thought that FRET-FLIM is independent from the concentration of the fluorochromes and that the increase in FRET efficiency is caused by a change in distance between the 2 reporter FP (CFP and YFP) and it would be possible to have less dimerisation of the proteins but an increase in FRET efficiency. Is this correct?
Thanks
Kees
Dr Ir K.R. Straatman
Senior Experimental Officer
Centre for Core Biotechnology Services
College of Medicine, Biological Sciences and Psychology
University of Leicester
http://www.le.ac.uk/biochem/microscopy/home.html
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Sydney, NSW 2006
Phone +61 2 9351 3176 Fax +61 2 9351 7682
Mobile 0413 281 861
______________________________________________
http://www.guycox.net
-----Original Message-----
From: Confocal Microscopy List [mailto:[log in to unmask]] On Behalf Of Straatman, Kees R. (Dr.)
Sent: Thursday, 17 November 2011 9:34 PM
To: [log in to unmask]
Subject: FRET-FLIM question
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Dear list members,
We have a discussion about FRET-FLIM at the moment and somebody has published that they see an increase in FRET efficiency and argues that this is because there is more dimerisation between the two proteins they are interested in. Now, I thought that FRET-FLIM is independent from the concentration of the fluorochromes and that the increase in FRET efficiency is caused by a change in distance between the 2 reporter FP (CFP and YFP) and it would be possible to have less dimerisation of the proteins but an increase in FRET efficiency. Is this correct?
Thanks
Kees
Dr Ir K.R. Straatman
Senior Experimental Officer
Centre for Core Biotechnology Services
College of Medicine, Biological Sciences and Psychology
University of Leicester
http://www.le.ac.uk/biochem/microscopy/home.html
-----
No virus found in this message.
Checked by AVG - www.avg.com
Version: 2012.0.1872 / Virus Database: 2092/4621 - Release Date: 11/16/11
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