Aptamer-Based Western Blot for Selective Protein Recognition.

Yao Wang,Zhe Li,Hanyang Yu

doi:10.3389/fchem.2020.570528

Abstract

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.

Highlights

Western blotting is a standard molecular biology technique for sensitive and selective protein detection (Nybo, 2012; Kim, 2017)
As alternative protein affinity reagents, hold great potential in saving both the time and the cost associated with Western blotting, we first attempted to leverage previously reported aptamer sequences and to assess their performances in Western blotting
The antibody labeled the target protein, and showed a number of non-specific bands (Figure 2B). These results suggested that using such selected DNA aptamers could achieve more selective protein detection in Western blotting in a cost-effective and timesaving fashion

Summary

Introduction

Western blotting is a standard molecular biology technique for sensitive and selective protein detection (Nybo, 2012; Kim, 2017). The common procedure of Western blotting analysis starts with electrophoresis separation of proteins followed by transfer to a nitrocellulose membrane. 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). Western blotting is very useful for protein identification and quantification, the whole procedure is laborious and time-consuming. To develop a simpler, more robust and affordable Western blotting technique that does not rely on antibodies would be useful to biomedical researchers

Methods

Results

Discussion

Conclusion