I want to transfect a shRNA-GFP against my protein of interest and observed how much this loss of function will affect the activity of other protein with a FRET ratiometric biosensor (CFP-YFP).
The ShRNA with GFP is to detect the transfected cells and evaluate FRET only in the positive GFP cells.
I will use a Zeiss 710 with the spectral separation.
Thanks in advance for any help
Hi,
GFP can be spectrally separated from CFP quite easily but will definitely bleedthrough into YFP channel. Therefore, your YFP/CFP ratio will be increased due to GFP emission and it might reduce your sensor dynamic range and sensitivity. In addition, comparison of cells with or without ShRNA will be problematic because of this. I would suggest to replace EGFP with mCherry or another fluorophore that could be easily separated from CFP/YFP.
Hope this helps,
Tal
Thank you, Tal, for your advice. I will choose another tool then.
Thanks again.