I have read several literatures regarding live cell FRET imaging and quantification over time. But is it possible to do it outside of cell, in vitro? I was recently told that it is possible but they could not produce an example. I tried to look for it online using every possible keyword but was unable to find something that convinced me about the possibility. Please enlighten me and suggest a literature of the same i anyone have the idea.
FRET can definitely be done with proteins in solution, although not by microscopy. Instead, you would use a cuvette-based spectrofluorimeter or fluorescence plate reader with a microtiter plate. This is a very standard method of doing enzyme assays or measuring the association of two molecules. A good example of its use in enzyme assays is for proteases or peptidases that cleave a peptide bearing a small molecule fluorescence donor and an acceptor. It should also work for measuring the dissociation constant of two interacting proteins, one of which acts as the donor and the other as the acceptor, as long as the distances involved are within an appropriate range for a reasonable energy transfer efficiency.
Here are a few examples.
Article Quantitative analysis of multi-protein interactions using FR...
https://pubmed.ncbi.nlm.nih.gov/34770748/
https://www.nature.com/articles/s41598-018-35535-9
There are many examples of in vitro FRET imaging in the literature, covering various applications and systems. Here are some of the recent publications that you might find relevant and interesting:
- 1: This paper describes the use of FLIM FRET imaging to measure the interaction between a fluorescently labeled antibody (trastuzumab) and its target receptor (HER2) in vitro and in vivo. The authors used a near-infrared (NIR) donor-acceptor pair (IRDye800CW and IRDye680RD) to minimize the background fluorescence and increase the tissue penetration depth. They showed that FLIM FRET imaging can quantify the binding of trastuzumab to HER2 in vitro using purified proteins and in vivo using tumor xenografts in mice.
- 2: This paper reports the use of intensity-based FRET imaging to monitor the conformational changes of a fluorescently labeled enzyme (glucose oxidase) in vitro. The authors used a green-red donor-acceptor pair (Alexa Fluor 488 and Alexa Fluor 594) to label the enzyme at two different sites. They showed that intensity-based FRET imaging can detect the changes in the FRET ratio as the enzyme undergoes different states of activity and inhibition in vitro.
- 3: This paper demonstrates the use of intensity-based FRET imaging to study the assembly of a fluorescently labeled protein complex (the 26S proteasome) in vitro. The authors used a blue-yellow donor-acceptor pair (CFP and YFP) to label the protein subunits of the complex. They showed that intensity-based FRET imaging can reveal the formation and dissociation of the proteasome complex in vitro under different conditions of ATP and salt.
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