I have been struggling with BiFC complementation assay. I have pBiFC VC155 and VN173 plasmids. I cloned my gene of interest and confirmed its expression in Western blot,but I could not see any fluorescence signal under fluorescence and confocal microscopy.I did Pull down assay to confirm its interaction inside the cell. What could be the reason?
I think there are two possibilities.
If you are sure that the interaction happens inside the cells you are using and that your constructs are expressing the POI-VC155 and POI2-VN173 fusion proteins, then the most reasonable cause is a microscopy issue. Are you sure you are using the right filters for visualizing the Venus protein? Can you visualize other fluorescent proteins with similar Ex/Em wavelengths?
Alternatively, did the pull-down assay identify interaction between the Venus-fused proteins, or only the endogenous ones? Same question for the Western blot: did you blot for the protein itself, or the fused versions?
If you only confirmed the interaction/expression of the non-fused versions, maybe the Venus domains inhibit the interaction and that is why you are not seeing fluorescence? (Would really be a case of bad luck.)
Let me know what you think.
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