Table 1. Binding Parameters of FREX Sensorsa.
| sensor variant | donor/acceptor type, location | donor, acceptor labeling efficiency (%) | Ka,app (M–1) | ratiometric response (x-fold change) |
|---|---|---|---|---|
| P48 | Alexa488/Alexa594b | 26, 109 | (2.41 ± 0.3) × 105 | 3.0 ± 0.2 |
| P60 | Alexa488/Alexa594b | 27, 106 | (7.30 ± 1) × 104 | 8.4 ± 0.9 |
| P69 | Alexa488/Alexa594b | 26, 106 | not detected | not detected |
| P48 | Alexa488 (Cys48)/Alexa594 (N-terminus) | 83, 107 | (3.15 ± 0.4) × 105 | 8.5 ± 0.9 |
| P60 | Alexa488 (Cys48)/Alexa594 (N-terminus) | 83, 106 | (5.62 ± 0.7) × 104 | 5.9 ± 0.3 |
| P48 (10% fetal bovine serum) | Alexa488/Alexa594b | 26, 109 | (1.22 ± 0.1) × 105 | 2.8 ± 0.1 |
| P48 (2:1 lysate ratio)c | CyPet/YPetb | not applicable | 1.7 × 105 | 1.3 |
| P48 (1:1 lysate ratio) | CyPet/YPetb | not applicable | 2.8 × 105 | 1.6 |
| P48 (1:2 lysate ratio) | CyPet/YPetb | not applicable | 2.6 × 105 | 1.8 |
a
Errors are standard deviations of three independent experiments.
b
The donor and acceptor are at N-termini.
c
Ratios reflect approximate molar ratios of CyPet-FN3BN+I75A to YPet-P48. Cell lysate volume ratios (CyPet-FN3BN+I75A:YPet-P48) are 1:1, 1:2, and 1:4 for the 2:1, 1:1, and 1:2 molar ratio samples, respectively.