XIE/2:2026/Jan

1.13

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.