Aptamer-Based Western Blot for Selective Protein Recognition.
Aptamer-Based Western Blot for Selective Protein Recognition.
复制标题
用于选择性蛋白质识别的基于适体的蛋白质印迹
DOI：
10.3389/fchem.2020.570528
复制
发表时间：
2020
影响因子：
5.5
通讯作者：
Yu H
中科院分区：
文献类型：
--
作者：
Wang Y;Li Z;Yu H
选择性蛋白质识别在分子生物学技术如Western印迹和ELISA中是至关重要的。在这些方法中，靶蛋白的成功检测依赖于抗体的特异性相互作用，这通常带来高的生产成本并且需要长的孵育时间。适体代表了用于蛋白质识别的另一类简单且负担得起的亲和试剂，并且在蛋白质印迹中用适体取代抗体可能更省时且更具成本效益。在这项工作中，多个荧光DNA适配体分离，通过体外选择性标记常用的标签蛋白，包括GST，MBP，和His标签。所产生的适体G1、M1和H1分别以纳摩尔亲和力特异性结合至其同源靶蛋白。与传统的基于抗体的免疫印迹相比，这种基于适体的方法提供了更清晰的背景，并且能够选择性地标记复杂混合物中的靶蛋白。最后，鉴定出的适体还可以有效识别具有相同标签的不同融合蛋白，从而大大扩展了这些适体的潜在应用范围。本工作为蛋白质印迹分析中选择性识别蛋白质提供了有用的分子工具。
Selective protein recognition is critical in molecular biology techniques such as Western blotting and ELISA. Successful detection of the target proteins in these methods relies on the specific interaction of the antibodies, which often bring a high production cost and require a long incubation time. Aptamers represent an alternative class of simple and affordable affinity reagents for protein recognition, and replacing antibodies with aptamers in Western blotting would potentially be more time- and cost-effective. In this work, multiple fluorescent DNA aptamers were isolated by in vitro selection to selectively label commonly used tag proteins including GST, MBP, and His-tag. The generated aptamers G1, M1, and H1 specifically bound to their cognate target proteins with nanomolar affinities, respectively. Compared with conventional antibody-based immunoblotting, such aptamer-based procedure gave a cleaner background and was able to selectively label target protein in a complex mixture. Lastly, the identified aptamers were also effective in recognition of different fusion proteins with the same tag, thus greatly expanding the scope of the potential applications of these aptamers. This work provided aptamers as useful molecular tools for selective protein recognition in Western blotting analysis.