Protocol 2: Difference between revisions
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== Protocol == | == Protocol == | ||
=== | === FRET Test=== | ||
*'''Stock Solution''': 1 mM | *'''Stock Solution''': 1 mM | ||
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*Fluorometer | *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) | |||
Revision as of 03:19, 15 December 2025
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)