Protocol 2: Difference between revisions
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* '''Antibody (lexatumumab)''': 50 μM stock; prepare 10 μM working solution by dilution in 1× PBS. | * '''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. | * '''TCEP''': 10 mM stock; prepare 100 μM working solution by dilution in nuclease-free water. | ||
* '''Mal-PEG4-DBCO''': 10 mM stock (DMSO); | * '''Mal-PEG4-DBCO''': 10 mM stock (DMSO); 100uM working solution (DMSO). | ||
* '''SsDNA-Azide''': 1 mM stock (nuclease-free water). | * '''SsDNA-Azide''': 1 mM stock (nuclease-free water); 100uM working solution (UP water). | ||
* '''Citrate buffer''': 100 mM, pH 5.5. | * '''Citrate buffer''': 100 mM, pH 5.5. | ||
=== 1 | === 1. Antibody reduction === | ||
* | * | ||
{| class="wikitable" | {| class="wikitable" | ||
| Line 31: | Line 14: | ||
! TCEP | ! TCEP | ||
! AB:TCEP | ! AB:TCEP | ||
! | ! PBS buffer (pH 6.5) | ||
|- | |- | ||
| 10 uL | | 10 uL | ||
| 5 uL | | 1.5 uL | ||
| 1:5 | | 1:1.5 | ||
| | | 88.5 uL | ||
|} | |} | ||
* Pre-dilute TCEP into | * 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. | * 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. | * Remove excess TCEP and exchange buffer to 1× PBS (pH 6.5) by ultrafiltration, performing four washes. | ||
=== | === 2. Antibody–Mal conjugation === | ||
* | * | ||
{| class="wikitable" | {| class="wikitable" | ||
! Antibody (R) | ! Antibody (R) | ||
! Mal | ! Mal | ||
! AB: | ! AB:Mal | ||
! 1X PBS (pH 6.5) | ! 1X PBS (pH 6.5) | ||
|- | |- | ||
| Line 56: | Line 39: | ||
|} | |} | ||
* Because Mal‑DNA contains DMSO (~10%), dilute the Mal‑DNA in PBS prior to addition to antibody. | * Because Mal‑DNA contains DMSO (~10%), dilute the Mal‑DNA in PBS prior to addition to antibody. | ||
* Incubate at '''room temperature 2 | * Incubate at '''room temperature 2 h ''' | ||
* Purify the conjugate by ultrafiltration/washing with 1× PBS (pH 7.4) four times to remove excess unreacted DNA. | * Purify the conjugate by ultrafiltration/washing with 1× PBS (pH 7.4) four times to remove excess unreacted DNA. | ||
=== 3. Antibody-DNA === | |||
* | |||
{| class="wikitable" | |||
! Mal-AB | |||
! SsDNA | |||
! Mal:DNA | |||
|- | |||
| ALL | |||
| 5 uL | |||
| 1:1 | |||
|} | |||
* Incubate the mixture at '''37℃ for 2 h'''. | |||
== Triplex DNA pH Test== | == Triplex DNA pH Test== | ||
Latest revision as of 06:24, 19 January 2026
Antibody-DNA Conjugate[edit]
Reagents and stock solutions[edit]
- 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); 100uM working solution (DMSO).
- SsDNA-Azide: 1 mM stock (nuclease-free water); 100uM working solution (UP water).
- Citrate buffer: 100 mM, pH 5.5.
1. Antibody reduction[edit]
| 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.
2. Antibody–Mal conjugation[edit]
| Antibody (R) | Mal | AB:Mal | 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 h
- Purify the conjugate by ultrafiltration/washing with 1× PBS (pH 7.4) four times to remove excess unreacted DNA.
3. Antibody-DNA[edit]
| Mal-AB | SsDNA | Mal:DNA |
|---|---|---|
| ALL | 5 uL | 1:1 |
- Incubate the mixture at 37℃ for 2 h.
Triplex DNA pH Test[edit]
- 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[edit]
- 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[edit]
- 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)