The fluorescent two-hybrid assay (F2H) is a technique for the validation and characterization of protein-protein interactions in vivo. It can be used for studying the modulation of interactions with inhibitors or activators, for compound screening and for confirmation of results from other protein-protein interaction screens such as co-immunoprecipitation studies.

In short, two proteins, bait and prey protein, are fused to GFP and RFP, respectively. A BHK cell line is transformed with ChromoTek’s F2H platform reagent and two DNA plasmids coding for the GFP-bait protein and the RFP-prey protein. The interaction of bait protein and prey protein can be detected by co-localization of a green and a red fluorescent spot in the nucleus of the BHK cells with an epi-fluorescence microscope.