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Hi Kees, 

FLIM data reports the change in the amount of donor energy transferred to the acceptor either per cell or per pixel.   One source of your problem could be that energy transfer always convolves the number of interacting proteins with the distance and alignment between donor and acceptor, both of which can change in a single experiment.   Most FRET studies try to simplify this question by assuming away the complexity - either dimers form of they do not (as in your colleague's argument), the complex is fixed and you measure its conformational change (e.g., most modern biosensors) or no binding is assumed and you are simply measuring the concentration of the two fluorophores (e.g., similar to fluorescence dequenching in liposome fusion).  You are asking whether the complexes can decrease in number while the remaining complexes change in conformation to bring the fluorophores much closer together.  This is possible in theory, but it would mean that the initial FRET had to be quite low and the loss of dimerization would have to be modest.  That kind of question is best tested using an independent measure of dimerization - you could try a co-IP, or else immobilize one binding partner with an antibody or cytoskeleton cross-linking domain and measure the mobility of the other using FRAP.  

Now I am confused by your description of BIFC data.  Bimolecular complementation is an irreversible reaction.  By 'goes down' do you mean that the rate of BIFC complex formation decreases, or do you really see a decrease in BIFC fluorescence when you add the third binding partner?  I would be concerned if the second case was true.  

cheers, 


TF

Timothy Feinstein, PhD
Postdoctoral Fellow 
Laboratory for GPCR Biology
Dept. of Pharmacology & Chemical Biology
University of Pittsburgh, School of Medicine 
BST W1301, 200 Lothrop St.
Pittsburgh, PA  15261

On Nov 17, 2011, at 7:51 AM, Straatman, Kees R. (Dr.) wrote:

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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
> To: [log in to unmask]
> 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
> ______________________________________________
> Associate Professor Guy Cox, MA, DPhil(Oxon) 
> Australian Centre for Microscopy & Microanalysis, 
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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
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> http://www.le.ac.uk/biochem/microscopy/home.html
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