Protocol 2: Difference between revisions

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)

@@ Line 1: / Line 1: @@
 <div style="color: black !important; font-family: 'Times New Roman', SimSun, serif !important, line-height:1.5;">
-== Protocol ==
+== Antibody-DNA Conjugate ==
-=== pH ===
+=== Reagents and stock solutions ===
-*'''Stock Solution''': 1 mM
+* '''Antibody (lexatumumab)''': 50 &mu;M stock; prepare 10 &mu;M working solution by dilution in 1× PBS.
+* '''TCEP''': 10 mM stock; prepare 100 &mu;M 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.
-*'''Buffer''': 0.5X TBE with 11 mM MgCl2 (pH: 5.5, 6.5, 7.0, 7.5, 8.5)
+=== 1. Antibody reduction ===
+*
+{| class="wikitable"
+! 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 ===
+*
+{| class="wikitable"
+! 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.
-*'''Incubation''': 37℃;C, 3 h
+=== 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==
+*'''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.
 {| class="wikitable"style="text-align:center; width:80%"
-! T-S1
+! pH (388 uL)
-! T-S2
+! SsDNA 1 (Cy3) — 4 uL
-! TFO
+! SsDNA 2 (Cy5) — 4 uL
-! Buffer
+! SsDNA 3 — 4 uL
-! pH
+|-
+| 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
 |-
-| 4 &mu;L
+| 7.5
-| 4 &mu;L
+| S-TFO
-| 4 &mu;L
+| S-S1
-| 388 &mu;L
+| S-S2
-| 5.5, 6.5, 7.0, 7.5, 8.5
+|-
+| 8.5
+| TFO
+| S1
+| S2
+|-
+| 8.5
+| S-TFO
+| S-S1
+| S-S2
 |}
-*Fluorometer
+=== 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)