Biochemical analyzes of endogenous argonaute complexes immunopurified with anti-Argonaute monoclonal antibodies.

Keita Miyoshi, Tomoko N. Okada, Haruhiko Siomi, Mikiko C. Siomi

研究成果: Article

4 引用 (Scopus)

抜粋

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.

元の言語English
ページ(範囲)29-43
ページ数15
ジャーナルMethods in molecular biology (Clifton, N.J.)
725
DOI
出版物ステータスPublished - 2011

ASJC Scopus subject areas

  • Molecular Biology
  • Genetics

フィンガープリント Biochemical analyzes of endogenous argonaute complexes immunopurified with anti-Argonaute monoclonal antibodies.' の研究トピックを掘り下げます。これらはともに一意のフィンガープリントを構成します。

  • これを引用