Hello,

I have a problem with protoplast transformation. I'm trying to conduct BiFC (Bimolecular fluorescence complementation) for checking interaction of two mitochondrial proteins. Beside test sample I have also positive control - two mitochondrial proteins which interaction was proven by BiFC in earlier publications. Protoplast are isolated from 4-week plants, after 1h digestion with cellulase (1.2% [m/v]) and macerase (0.4% [m/v]) protoplasts were washed 3 times in W5 buffer, and resuspended with MMg solution. Protoplasts were transformed with 5 ug of each construct in 20% PEG (P3640). After collecting protoplasts with centrifugation they were resuspended with W1 solution and kept in dark overnight (22°C). On the confocal microscope I was expecting signal when 514 nm laser was used (EYFP excitation length), but no such signal was obtained, either from positive control or tested samples. Protoplasts were intact after overnight incubation, but transformation is probably not working. Am I doing something wrong?