**4.2. Stability of probes in vitro**

The same Fluc derived from *P. pyralis* was applied to both Fluc-based PCA in vitro and FlimPIA. Then, the thermostability of probes was compared [10]. The probes of Fluc-based PCA (FKBP-C and FRB-N) and the probes of FlimPIA (FKBP-Donor and FRB-Acceptor) were preincubated with or without rapamycin at 37°C (**Figure 10A**). After 30 minutes, half of the luminescence signal was retained in FlimPIA, and on the other hand, the luminescence signal was almost completely diminished in PCA. The rate of the luminescence decay in FlimPIA was approximately one-fourth of the rate of the decay in PCA (**Figure 10B**).

**4.4. Sensitivity in vitro**

Fluc-based PCA.

The detectable limits of the concentration of rapamycin in Fluc-based PCA, the conventional FlimPIA, and the improved FlimPIA were compared, when 50 nM of each probe (FKBP-C and FRB-N, or FKBP-Donor and FRB-Acceptor) and rapamycin were used. The limits were 250 pM in Fluc-based PCA and 10 pM in FlimPIA using the K529Q/S440L mutant as the Acceptor [4, 9, 10]. The sensitivity of the improved FlimPIA was higher than the sensitivity of

**Figure 10.** Comparison of thermostability in vitro. (A) Probes (50 nM each) were preincubated at 37°C with or without

FlimPIA, Right: Fluc-based PCA. Red: incubation for 0 min with rapamycin, Orange: 15 min with rapamycin, pink: 30 min with rapamycin, green: 0 min without rapamycin, light blue: 15 min without rapamycin, dark blue: 30 min without rapamycin. (n = 3) (B) Inactivation time course. Relative luminescent intensities at 4 s after reaction start were

and ATP). Left:

equimolar rapamycin. The luminescent intensity was measured for 4 s after adding substrates (LH<sup>2</sup>

normalized at the value obtained with 0 min pre-incubation (n = 3). ©American Chemical Society.

**Figure 9.** FKBP12-FRB association detected by FlimPIA in cultured cells. ©American Chemical Society.

A Novel Protein-Protein Interaction Assay Based on the Functional Complementation of Mutant…

http://dx.doi.org/10.5772/intechopen.75644

23
