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.

Western blot for protein quantification