Abstract
Argonaute proteins are key factors in RNA silencing. After association with small RNAs of 20–30 Ânucleotides, Argonaute proteins are targeted to homologous RNA molecules that are to be silenced. To understand the functional contributions of Argonaute proteins to RNA silencing at a biochemical level, immunoisolation of Argonaute proteins from living cells of various organisms has been performed. This has enabled the analysis of Argonaute-associated proteins and RNAs. Identifying the small RNAs that associate with individual Argonaute proteins, for instance, could help to elucidate the silencing pathways in which particular Argonaute proteins are involved. However, it is also necessary to note that the results obtained through such biochemical analyzes are greatly affected by the quality and properties of the antibodies used, as well as by the immunoprecipitation conditions employed, including buffer contents and/or salt concentration. In this chapter, we describe fundamental methods for immunoprecipitating Argonaute proteins using monoclonal antibodies as well as for detecting associated proteins and small RNAs. Furthermore, we will also explain how various parameters, such as antibody properties and buffer conditions, can alter the production and interpretation of experimental data.
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Acknowledgments
We thank Asuka Azuma-Mukai for technical assistance and other members of the Siomi laboratory for comments and for critical reading of the manuscript. This work was supported by Mochida Memorial Foundation for Medical and Pharmaceutical Research grants to K.M., MEXT grants to H.S. and NEDO (New Energy and Industrial Technology Development Organization) grants to M.C.S. M.C.S. is supported by CREST from JST.
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Miyoshi, K., Okada, T.N., Siomi, H., Siomi, M.C. (2011). Biochemical Analyzes of Endogenous Argonaute Complexes Immunopurified with Anti-Argonaute Monoclonal Antibodies. In: Hobman, T., Duchaine, T. (eds) Argonaute Proteins. Methods in Molecular Biology, vol 725. Humana Press. https://doi.org/10.1007/978-1-61779-046-1_3
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DOI: https://doi.org/10.1007/978-1-61779-046-1_3
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