Protocol 2
Antibody-DNA Conjugate
Reagents and stock solutions
- Antibody (lexatumumab): 50 μM stock; prepare 10 μM working solution by dilution in 1× PBS.
- TCEP: 10 mM stock; prepare 100 μM working solution by dilution in nuclease-free water.
- Mal-PEG4-DBCO: 10 mM stock (DMSO); 1 mM working solution (DMSO).
- SsDNA-Azide: 1 mM stock (nuclease-free water).
- Citrate buffer: 100 mM, pH 5.5.
1. Preparation of Mal–DNA
| Mal-PEG4-DBCO | SsDNA | Mal:DNA | 1X PBS (pH 6.5) | Concentration |
|---|---|---|---|---|
| 2 uL | 4 uL | 1:2 | 14 uL | 100 uM |
- Incubate the mixture at 37℃ for 2 h.
1. Antibody reduction
| Antibody | TCEP | AB:TCEP | PBS buffer (pH 6.5) |
|---|---|---|---|
| 10 uL | 1.5 uL | 1:1.5 | 88.5 uL |
- Pre-dilute TCEP into buffer (pH 6.5) before mixing with antibody to avoid antibody inactivation (TCEP stock is acidic).
- Incubate at 37 ℃ for 2 h to reduce disulfides and generate free thiols.
- Remove excess TCEP and exchange buffer to 1× PBS (pH 6.5) by ultrafiltration, performing four washes.
3. Antibody–Mal conjugation
| Antibody (R) | Mal | AB:DNA | 1X PBS (pH 6.5) |
|---|---|---|---|
| All | 5 uL | 1:5 | 50 uL |
- Because Mal‑DNA contains DMSO (~10%), dilute the Mal‑DNA in PBS prior to addition to antibody.
- Incubate at room temperature 2.5 h
- Purify the conjugate by ultrafiltration/washing with 1× PBS (pH 7.4) four times to remove excess unreacted DNA.
Triplex DNA pH Test
- Stock Solution: Dissolve in ultrapure water to 1 mM and store at −20 °C.
- Buffer: 0.5X TBE with 10 mM MgCl2 (pH: 5.5, 6.5, 7.0, 7.5, 8.5)
- Incubation: Dilute the stock with ultrapure water to 1% (10 µM). Add 4 µL each of TFO, S1, and S2 (or S‑TFO, S‑S1, S‑S2) to 388 ul buffers of different pH and incubate at 37 °C for 3 h.
| pH (388 uL) | SsDNA 1 (Cy3) — 4 uL | SsDNA 2 (Cy5) — 4 uL | SsDNA 3 — 4 uL |
|---|---|---|---|
| 5.5 | TFO | S1 | S2 |
| 5.5 | S-TFO | S-S1 | S-S2 |
| 6.5 | TFO | S1 | S2 |
| 6.5 | S-TFO | S-S1 | S-S2 |
| 7.0 | TFO | S1 | S2 |
| 7.0 | S-TFO | S-S1 | S-S2 |
| 7.5 | TFO | S1 | S2 |
| 7.5 | S-TFO | S-S1 | S-S2 |
| 8.5 | TFO | S1 | S2 |
| 8.5 | S-TFO | S-S1 | S-S2 |
Fluorometer
- Donor-only:Cy3-only
- Acceptor-only:Cy5-only
- Measurement channels
- IDA channel: excitation 550 nm; emission 670 nm.
- IDD channel: excitation 550 nm; emission 570 nm.
- IAA channel: excitation 650 nm; emission 670 nm.
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)