XIE/2:2026/Jan

1.13

Synthesis of Anti-Oligo

抗体还原

• 取2 ul(1mM) TCEP,用 PBS(7.4) 稀释至 20 ul(100uM),取 5 ul稀释后的 TCEP ，加入 85 ul PBS(7.40)，涡旋混匀---TCEP溶液pH过低提前中和
• 取2 ul(50uM)抗体，用 PBS（pH 7.4）稀释至 10 ul（10uM），将 TECP 的 PBS 缓冲液加入其中，37℃ 孵育 2 h.
• 取1 ul(50uM)抗体，用 PBS（pH7.4）稀释至 8 ul，作为 C 组

偶联

• 将孵育好的溶液用 PBS(6.5) 洗涤4次（10000g 3.5min），均分样品（A.B）----buffer pH 6.5避免后续加入的Mal被水解（碱性易水解）
• A（共混组）：向 A 中加入 5 ul(100uM) 的 Mal-PEg-DBCO 和 1 ul(1mM) Azide-ssDNA，室温避光孵育 4 h.
• B（依次组）：1）向 B 中加入 5 ul(100uM) 的 Mal-PEg-DBCO，室温避光孵育 2 h. 2)孵育结束后，向其加入1 ul(1mM) Azide-ssDNA，室温避光孵育 2 h.

验证

• 强还原断开轻重链：向A、B、C中加入对应量的蛋白 loudingbuffer ，再各加入1 ulβ-巯基乙醇，100℃煮 10m in
• 10% SDS-Page.

1.19

incubation

• DNA samples were stratified by thiolation status into two primary groups: thiolated DNA (bearing a thiol modification) and non-thiolated DNA (without thiol modification). Within each primary group, samples were further subdivided by phosphate-buffered saline (PBS) pH, yielding subgroups at pH 5.5, 6.5, 6.7, 6.8, 7.0, 7.5, and 8.5. PBS solutions were prepared and adjusted to the target pH and verified with a calibrated pH meter at room temperature.

S1 (100 μM)	S2 (100 μM)	TFO (100 μM)	Buffer
2 μl	2 μl	2 μl	194 μl

FRET

• Method：Samples were measured in Cy3 and Cy5 channels using appropriate excitation/emission filters under constant instrument settings.

1.20

Antibody reduction

1. Dilute 2 µL of antibody stock with 1× PBS to 10 µL (10 µM).
2. Prepare a TCEP working solution by diluting 1.5 µL of 100 µM TCEP with 1× PBS to 90 µL.
3. Mix the 10 µL antibody with 90 µL TCEP working solution and incubate at 37°C for 2.5 h (final antibody ≈1 µM; TCEP ≈1.5 molar equivalents).
4. Purify the reduced antibody by three rounds of buffer exchange into 1× PBS.

Antibody functionalization with DBCO

• Add 5 µL of Mal‑PEG4‑DBCO (100 µM) to the purified, reduced antibody and incubate at 37°C for 2 h (5 eq).

Antibody conjugation to oligo

• Add 5 µL of Azide‑Oligo (100 µM) to the DBCO‑modified antibody and incubate at 37°C for 2 h to complete the strain‑promoted azide‑alkyne cycloaddition (SPAAC) coupling (5 eq).

Native-page

• Analyze reaction products by non‑denaturing electrophoresis using 7.5% native PAGE.
• Marker、Control（antibody）、Antibody-Oligo、Antibody-DNA、Marker

Conclusion :Nativepage is not a good chose antibody conjugation

1.21

Native-page

• Samples(1.20 antibody with oligo) and controls were analyzed by native polyacrylamide gel electrophoresis (PAGE) using a 3–5% stacking gel and a 12.5% resolving gel.
• Marker、Control（antibody）、Antibody-Oligo、Antibody-DNA、Marker

1.22

1.28

抗体偶联

平均偶联数量测量

• 双波长法计算标记度（Degree of Labeling, DoL）

以下公式采用 A280 和 A260 的吸光度值，通过校正 DNA 在 280 nm 处的贡献来计算抗体浓度、核酸浓度及最终的平均偶联数（DoL）。

1. CAb = [A280 − CF_DNA,280·A260] / εAb,280·L
2. CDNA = A260 / εDNA,260·L
3. DoL = CDNA / CAb、

• L 光程 这里为 1；A280，A260 样品吸光度；
• CF_DNA,280·A260 “纯DNA”在同缓冲下测得的比值 A280/A260，用来估算DNA在280的交叉吸收；
• εAb,280 抗体消光系数，本抗体为2.3865×10^5 M⁻¹·cm⁻¹；
• εDNA,260 Oligo消光系数，该Oligo(TTTTCTCCTCTCTCCTCCTCTT，5'Azide)为 1.698×10^5 M⁻¹·cm⁻¹；

SDS-page验证偶联结果