I'm using the Operetta system to measure the FRET signal between two proteins in the Golgi (tags are YFP and mCherry). The majority of the signal comes from protein crowding and not from real protein-protein interaction, which makes the technique only useful for comparative experiments. I tried to make a control where I measured the FRET signal between two proteins that are known not to interact. The FRET signal was still high and a positive interaction is only 5 to 20% higher, which makes the window of difference and the FRET sensitivity low and how can I solve this problem?
Do you think it would help if you used a donor-acceptor pair with a shorter Forster distance?
How are you expressing the protein? Transient transfection? Maybe you could try a weaker promoter so your proteins of interest aren't in such high abundance?
You could also CRISPR modify the endogenous loci to include florescent tags so you are monitoring physiologically accurate protein levels.
Thank you for your answers.
Dr. Shapiro do you mean a donor and acceptor with a smaller size? because it might be that the smaller they are the shorter distance they need for energy transfer. I've found an interesting donor-acceptor in this publication: http://www.pnas.org/content/106/38/16227.full
BSc. Sharma, I'm doing transient transfections and not willing to go into CRISPR and stable cell lines, I have a lot of mutations and chimeric proteins to test and it will take forever. I will however try to express the proteins with a shorter time and at a lower temperature (30 degrees Celsius) to see if I can get better positive signal.
My question was not about the size of the donor and acceptor molecules. It was about R0, the distance between donor and acceptor at which the efficiency of energy transfer is 50%. This is a characteristic of the donor-acceptor pair.
Thanks Dr. Shapiro for your clarification, I've found an article in which they describe the FRET efficiency and the R0 for different pairs. The difference in R0 between mVenus, mCherry pair and the pair with lowest R0 is around 1 to 1,5nm. Which might enhance the specificity but not by much. However, they mentionned that the mCherry suffers from low brightness and the FRET emission is too weak to detect above the donor emission tail. Which explains why I see low mCherry and low FRET intensity when compared to mVenus intensity.
Bottom line: The lab used before the CFP, YFP pair in Flow Cytometry and Acceptor Photobleaching experiments and it worked better. So we're going to test this pair in Operetta. The reason why we switched to mVenus, mCherry pair is because it was advised that they work better with the filters we have. We'll also reduce the concentration of DNA, the culture time and the temperature and see how it goes.
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