David M Jameson - I am aiming to create fractions corresponding to nuclear, mitochondrial, cytoplasmic, and ER/Golgi from human cells for use in Western blotting and co-IP.
Well a key consideration is choosing your donor and acceptor Fluorescent Proteins properly. I presume you will be doing FLIM measurements to watch the donor lifetime decrease during FRET? What type of microscope do you have? Will you be doing two-photon excitation?