![]() ![]() First, aptamers can be generated to bind protein targets that are poorly immunogenic or toxic to animals. Compared with antibodies, aptamers possess several advantageous features as protein affinity reagents ( Groff et al., 2015 Crivianu-Gaita and Thompson, 2016 Kooshki et al., 2019). Aptamers have been generated by in vitro selection to recognize a wide variety of targets ranging from metal ions, small organic compounds and biomacromolecules ( Tuleuova et al., 2010 Sharma et al., 2012 Qu et al., 2016 Ruscito and DeRosa, 2016) to viruses, bacteria and human cells ( Medley et al., 2011 Wandtke et al., 2015 Marton et al., 2016 Wang et al., 2018). Therefore, to develop a simpler, more robust and affordable Western blotting technique that does not rely on antibodies would be useful to biomedical researchers.Īptamers are short single-stranded oligonucleotides that bind their targets with high affinity and specificity by forming distinct tertiary structures ( Ellington and Szostak, 1990 Robertson and Joyce, 1990 Tuerk and Gold, 1990 Wang et al., 2019). In addition, antibodies as the key affinity reagent usually suffer from high production cost and are prone to irreversible denaturation ( Groff et al., 2015). Although Western blotting is very useful for protein identification and quantification, the whole procedure is laborious and time-consuming. After blocking for non-specific adsorption, specific protein recognition is achieved by interaction with an appropriate primary antibody and secondary antibody, which is often conjugated to a fluorophore or an enzyme such as horseradish peroxidase ( Mishra et al., 2019 Han et al., 2020). The common procedure of Western blotting analysis starts with electrophoresis separation of proteins followed by transfer to a nitrocellulose membrane. Western blotting is a standard molecular biology technique for sensitive and selective protein detection ( Nybo, 2012 Kim, 2017). This work provided aptamers as useful molecular tools for selective protein recognition in Western blotting analysis. 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. 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. The generated aptamers G1, M1, and H1 specifically bound to their cognate target proteins with nanomolar affinities, respectively. 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. 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. 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. Selective protein recognition is critical in molecular biology techniques such as Western blotting and ELISA. 4State Key Laboratory of Analytical Chemistry for Life Science, Nanjing University, Nanjing, China.3Chemistry and Biomedicine Innovation Center (ChemBIC), Nanjing University, Nanjing, China.2State Key Laboratory of Coordination Chemistry, Nanjing University, Nanjing, China.1Department of Biomedical Engineering, College of Engineering and Applied Sciences, Jiangsu Key Laboratory of Artificial Functional Materials, Nanjing University, Nanjing, China.Yao Wang 1,2,3 Zhe Li 1,4 Hanyang Yu 1,2,3 * ![]()
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