One challenge facing BiFC users is the difficulty in identifying appropriate negative controls (Figure 4, B and C). This is often the case when the two proteins are new and no prior biochemical or structural information is available. To overcome this challenge, BiFC competition analysis should be considered as an alternative approach (Figure 5). The methodology behind BiFC competition analysis was originally reported using purified bJun-YN155 and bFos-YC155 fusion proteins (8). When differing amounts of free bJun were added to the assay, fluorescence complementation was inhibited in a dose-dependent manner (8) (Figure 5). The competitive nature of bJun interactions was also demonstrated using the multicolor BiFC assay to analyze interactions among bJun, bFos, and bATF2 (15). BiFC competition analysis has been successfully used in other BiFC experiments as well (17, 19, 62). However, it is important to note that the competitor must be co-expressed with the two fusion proteins, if not before the expression of the two fusions, since the BiFC complex is essentially irreversible once formed (8, 33, 43, 48-51). As demonstrated in the same bJun-YN155/bFos-YC155 system, addition of bJun at later times failed to efficiently inhibit fluorescence complementation (8).
In summary, we strongly recommend the use of a mutant control where a single mutation or small deletion is introduced into the interaction interface in one of the two interacting proteins. If such a mutant control is not available, one should at least perform BiFC competition analysis. In either case, quantification of the BiFC signal is desired unless all-or-none results are obtained.
Here we have summarized the recent development of BiFC technology and provided a perspective on future developments. Since the first use of BiFC for visualization of PPIs in mammalian cells a decade ago, various BiFC systems and applications have been reported for use in a wide range of cells and model organisms. Continued development of new BiFC systems will likely further expand and enhance applications to biological research.
The authors thank Holli Duren for technical assistant regarding BiFC experiments in C. elegans. The authors also thank Toyobo Biotechnology Foundation (long-term research grants) for support of collaboration between Y.K. and C.D.H.
The authors declare no competing interests.
Correspondence Address correspondence to Yutaka Kodama, Center for Bioscience Research and Education, Utsunomiya University, Tochigi, Japan. Email: [email protected]">[email protected] or Chang-Deng Hu, Department of Medicinal Chemistry and Molecular Pharmacology and Purdue University Center for Cancer Research, Purdue University, West Lafayette, IN, USA. Email: [email protected]">[email protected]References
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