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生物素标记EMSA探针是针对 EMSA 电泳条带的特点采用双端多重标记研发制备的高灵敏度标记探针。EMSA 实验由于多种原因，电泳条带普遍载量少，信号弱，如探针标记不佳，常常会出现假阴性检测结果，导致实验失败。本产品采用双端多重标记技术，对 EMSA 探针进行高效生物素标记，标记的探针阳性信号更强，可大幅提升 EMSA 后期 WB 的检测灵敏度，提高 EMSA 实验的成功率。
p53 consensus oligo的序列如下：
5'-TAC AGA ACA TGT CTA AGC ATG CTG GGG ACT-3'
3'-ATG TCT TGT ACA GAT TCG TAC GAC CCC TGA-5'
本探针采用生物素标记，免除放射性的危险与处理同位素废物的麻烦，减少环境污染，非常便于使用。生物素标记EMSA探针已经过纯化，可以直接用于 EMSA 结合反应。
产品特点：
1. 采用生物素标记，无放射性污染；
2. 双端生物素多重标记，阳性信号更强，可大幅提升检测灵敏度，提高EMSA 实验成功率；
3. 标记 EMSA 探针已经过纯化，可以直接用于 EMSA 结合反应。
操作步骤：
1. 可参考下表设置 EMSA 反应体系。
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2. 按照上述顺序依次加入除了生物素标记EMSA探针的其他组分，混匀，并且室温 (20-25℃) 放置 10 分钟，从而消除可能发生的探针和蛋白的非特异性结合，或者让冷探针优先反应。然后加入生物素标记EMSA探针，混匀，室温 (20-25℃) 放置 20 分钟。
3. 加入 1μl 10×EMSA 上样缓冲液，混匀后立即上样。注意溴酚蓝可能会影响蛋白和 DNA 的结合，建议尽量使用无色的上样缓冲液。如果使用无色上样缓冲液在上样时感觉不方便，可以在无色上样缓冲液里面添加极少量的蓝色上样缓冲液，至可以观察到蓝颜色即可。
注意事项：
1. 避免加热到 40℃以上，温度过高会导致双链 DNA 探针解聚成单链。单链无法用于 EMSA 研究。
2. 一个包装的生物素标记探针可以进行约 200-400 个样品的 EMSA 检测。
3. 做 EMSA 实验前可用斑点杂交验证探针标记效果。直接吸 1-2μL 标记探针滴加到裁剪浸泡好的尼龙膜上，室温干燥 30min，120℃烘烤固定 30min 后，用生物素标记核酸探针检测试剂盒检测即可。
4. 标记探针的用量对结果影响很大，过多或过少均会影响实验结果，研究者可先使用 0.5μL、1.0μL、1.5μL 进行优化。
6. 注意个人防护，请穿实验服并戴一次性手套操作。仅用于科研实验，勿用于临床诊断。
Biotin-Labeled EMSA Probe is a highly sensitive labeled probe developed and prepared using double-ended multiplex labeling, tailored to the characteristics of EMSA electrophoresis bands. EMSA assays often suffer from low loading amounts and weak signals of electrophoresis bands due to various factors. Poor probe labeling frequently causes false-negative results and experimental failure. This product adopts dual-terminal multiple labeling technology for efficient biotin labeling of EMSA probes. The labeled probe produces stronger positive signals, which greatly improves the detection sensitivity in the subsequent EMSA blotting step and raises the success rate of EMSA experiments.
The sequence of p53 consensus oligo is as follows:
5'-TAC AGA ACA TGT CTA AGC ATG CTG GGG ACT-3'
3'-ATG TCT TGT ACA GAT TCG TAC GAC CCC TGA-5'
Labeled with biotin, this probe eliminates radiation hazards and the hassle of handling radioactive waste, reduces environmental pollution, and is user-friendly. Biotin-Labeled EMSA Probe is pre-purified and can be directly applied to EMSA binding reactions.
Product Features:
1. Biotin labeling without radioactive contamination.
2. Dual-terminal multiple biotin labeling delivers stronger positive signals, substantially enhances detection sensitivity and improves EMSA experimental success rate.
3. Pre-purified labeled EMSA probe, ready for direct use in EMSA binding reactions.
Protocol:
1. Prepare EMSA reaction systems referring to the table below.
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2. Add all components except Biotin-Labeled EMSA Probe in the order listed above, mix well, and incubate at room temperature (20–25 °C) for 10 min. This step minimizes non-specific protein–probe interactions or allows cold probe to bind preferentially. Then add Biotin-labeled EMSA Probe, mix thoroughly, and incubate at room temperature (20–25 °C) for 20 min.
3. Add 1 μL of 10× EMSA Loading Buffer, mix and load immediately. Bromophenol blue may interfere with protein-DNA binding; colorless loading buffer is recommended. If colorless loading buffer is difficult for sample loading, a tiny amount of blue loading buffer can be added until a faint blue color is visible.
Precautions:
1. Do not heat above 40 °C. Excessively high temperature denatures double-stranded DNA probes into single strands, which are not suitable for EMSA analysis.
2. One package of biotin-labeled probe supports approximately 200–400 EMSA samples.
3. Dot blot can be used to verify labeling efficiency prior to EMSA. Spot 1–2 μL labeled probe onto pre-wetted nylon membrane, air-dry at room temperature for 30 min, bake at 120 °C for 30 min for fixation, then detect using a biotin-labeled nucleic acid probe detection kit.
4. The amount of labeled probe significantly affects results. Too much or too little probe will compromise experimental outcomes. Researchers may optimize dosage using 0.5 μL, 1.0 μL and 1.5 μL.
5. Wear lab coat and disposable gloves for personal protection. For research use only; not for clinical diagnosis.
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