Protocol 2
Protocol
FRET Test
- Stock Solution: 1 mM
- Buffer: 0.5X TBE with 10 mM MgCl2 (pH: 5.5, 6.5, 7.0, 7.5, 8.5)
- Incubation: 37℃;C, 3 h
| T-S1 | T-S2 | TFO | Buffer | pH |
|---|---|---|---|---|
| 4 μL | 4 μL | 4 μL | 388 μL | 5.5 , 6.5 , 7.0 , 7.5 , 8.5 |
- Fluorometer
Formuals
- α = mean(IDA_Donly / IDD_Donly)
(donor → acceptor leakage ratio)
- β = mean(IDA_Aonly / IAA_Aonly)
(acceptor direct‑excitation by the donor excitation ratio)
- IDA_corr = IDA_meas - α*IDD_meas − β*IAA_meas
(If the result < 0, set it to 0 .)
- Eapp = IDA_corr / (IDA_corr + γ * IDD_meas), here γ ≈ 1 (approximate)