I am trying to do FRET with CFP-YFP pair. I am getting FRET efficiency in the CFP-construct with YFP vector only pair as well. I am using TM FV 1000 con focal laser scanning microscopy. I don't know whether I am doing anything wrong. Would you please help me for trouble shooting?
How are you calculating FRET efficiency? Some direct excitation of YFP is always going to occur. In many systems, acceptor photobleaching is a better measure of specific FRET signal (i.e. measuring gain of signal in CFP channel after YFP photobleaching). Have you tried this?
Daniel, I am using acceptor photobleaching only.and then checking gain of signal in CFP channel.
Goutam- and do you see the same recovery of signal in both your YFP-tagged gene of interest and an untagged YFP control? FRET is a difficult business, especially because it is strongly affected by orientation of the chromophores, accidental photobleaching of your samples, and a small dynamic range of change in FRET efficiency (among other problems).
What other evidence do you have that there is a protein-protein interaction in your system, independent of FRET? While there are any number of reasons a FRET probe might fail, the most obvious is that the interaction is not present, or is too weak/transient to detect by this method.
Are you working in vitro, in cellulo, or in vivo? What cell type?
Daniel.. ya its very difficult business..initailly I was doing Live Cell system but there was some problem known interacting partner was not giving FRET signal so. I tried this fixed sample in that confocal microscope. and in this fixed sample its shwoing FRET positive in the vector alone as well.
I have many other evidences, I have done GST Pull down, Co-IP, and also co-localization. all are nicely giving interaction...here I was doing in cells, I have used both HEK 293T cells and Neuro2A cells.
In my experience, fixed cells never work for FRET, largely because of photobleaching. I've worked with reasonably low tech setups (heated microscope, CO2 independent media) and had good success.
If you're working with 293T cells, then your expression will likely be extremely high. High enough that you can simply make a lysate (cell-free system), and interrogate your FRET on a fluorimeter. In my experience, this will be the most quantitative and sensitive approach.
Hi Francois.. Thanku very much this is very interesting suggestion...But I wll say this s very tricky experiment...I was trying to do FRET by Live Cell in a nikon microscope I dnt know Iw as not successful. there even two known interactiong proteins didnt give signal and when I did the same experiment in the fixed cells I got signal in the vector control also..strange but true....lets see.many thanks again.
Goutam- sometimes the FRET tags themselves can interfere with protein-protein interactions, and also orientation of the tags plays a big role in FRET efficiency. You should confirm the interaction of your two "known" binding partners by co-IP (tagged versions) before you make any conclusions about how to interpret the FRET results.
Hi Daniel, you meant I should do the Co-IP with the same interacting molecules with the same tagging as in FRET experiment.. That I have not done actually.. well that I can do first...Yes... thank you very much Daniel.
Yes, the co-IP is important to show that the tags are not interfering with the protein-protein interaction.
In attached paper is example of FRET between fluorescent protein and conducting polymer. With experimental protocol included
Article Immunosensor based on fluorescence quenching matrix of the c...
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