In single molecular FRET, a GEF protein with N-terminal YFP and C-terminal CFP gave nice YFP and slightly lower CFP signal. The same protein with the YFP tag on either N or C-terminal gave less fluorescence than the partner carrying CFP. The problem is the GEF with YFP tag, though very well expressed gives less fluorescence. Do you have any ideas how to improve the fluorescence?
I am assuming that these are live cell experiments. First of all, is your excitation intensity for CFP (in the CFP-YFP experiment) same as the excitation intensity for the YFP only experiment? You can measure the laser power at the sample and find out how many watts/cm2 are you getting in each case. Secondly, I would give sufficient time to YFP for maturation. While the reported numbers vary, it may be a good idea to monitor if the YFP signal goes up as a function of time in the YFP only experiment. For me try between 10-60 mins. Finally, make sure that this high/ low/ slightly lower signals are actually quantifiable and not just some artifact of the camera. Hope this helps. Best of luck!
@Saurabh and Fernandes. I use a single wavelength LED (440 nm) to excite CFP and takes the YFP and CFP using a beam splitter in front of the camera. the setup works very well for all other FRET sensors. So, I am sure technically its fine. But, somehow the GEF protein of my interest, which is attached to YFP hinder the YFP fluorescence. I would like to know whether you have any ideas to increase the YFP signal.
- Badges
- Science topic
- Similar topics
- Physical Sciences
- Physical Phenomena
- Luminescence
- Fluorescence