**Author details**

**4.5. Detection limit of dimension of interacting protein in vitro**

Acceptor (left) (n = 3). ©American Chemical Society.

24 Protein-Protein Interaction Assays

determines the detectable dimensions of the interacting protein.

A fundamental limitation of FRET is that the detectable distance between the two probes is less than several nanometers, because the fluorescent signal is inversely proportional to the sixth power of the distance. A part of fibronectin type III, the seventh and eighth domains (Fn7-8), has a rigid structure with a 7 nm N-C terminal distance [10]. Ohashi et al. reported that a FRET signal using YPet and CyPet could not be observed by inserting Fn7-8 between the two fluorescent proteins [22]. The limit of the detectable distance between the two probes

**Figure 11.** Detectable distance between the probes in vitro. (A) Scheme of the assays. A long (7 nm) Fn7-8 domain is inserted between a binding domain (FKBP12) and a probe. Signals with and without equimolar rapamycin were compared. (B) FRET using 40 nM each of FKBP-Fn7-8-Cerulean and the FRB-YPet as a probe pair. (C) Fluc-PCA using 100 nM each of FKBP-Fn7-8-C and FRB-N (n = 3). (D) FlimPIA using 100 nM each of FKBP-Fn7-8-Donor and FRB-

Therefore, we compared the limit of the detectable distance between the probes in our assay. To examine this, Fn7-8 was inserted between FKBP12 and one of the probes (C-terminal domain for PCA, cerulean for FRET, and Donor for FlimPIA) (**Figure 11**). The large probes Yuki Ohmuro-Matsuyama and Hiroshi Ueda\*

\*Address all correspondence to: ueda@res.titech.ac.jp

Laboratory for Chemistry and Life Science, Institute of Innovative Research, Tokyo Institute of Technology, Yokohama, Japan
