title: Biochemical analyzes of endogenous argonaute complexes immunopurified with anti-Argonaute monoclonal antibodies.
abstract: Argonaute proteins are key factors in RNA silencing. After association with small RNAs of 20-30 -nucleotides, 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.
authors.affiliation: Department of Molecular Biology, Keio University School of Medicine, Tokyo, Japan.
authors.forename: Keita
authors.initials: K
authors.lastname: Miyoshi
authors.forename: Tomoko N
authors.initials: TN
authors.lastname: Okada
authors.forename: Haruhiko
authors.initials: H
authors.lastname: Siomi
authors.forename: Mikiko C
authors.initials: MC
authors.lastname: Siomi
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000378:metabolism*; D060565:Argonaute Proteins; D002460:Cell Line; D004330:Drosophila / Q000235:genetics / Q000378:metabolism; D029721:Drosophila Proteins / Q000235:genetics / Q000378:metabolism*; D047468:Immunoprecipitation; D035683:MicroRNAs / Q000235:genetics / Q000378:metabolism; D034743:RNA-Induced Silencing Complex / Q000235:genetics / Q000378:metabolism*
chemical_list: C433807:AGO2 protein, Drosophila; D000911:Antibodies, Monoclonal; D060565:Argonaute Proteins; D029721:Drosophila Proteins; D035683:MicroRNAs; D034743:RNA-Induced Silencing Complex