title: Loading of Argonaute Protein with Small Duplex RNA in Cellular Extracts.
abstract: Argonaute (Ago) proteins are the minimum core proteins required for executing RNA interference (RNAi) mechanisms of gene regulation. For Ago proteins to regulate gene expression through RNAi they must be loaded, or "programmed," with a single strand of small RNA. Natural small RNAs are typically double-stranded duplexes that require additional factors for efficient and specific loading into Ago proteins. Here, a protocol is described for investigating RNAi programming through loading of human Ago2 using radiolabeled small interfering RNA (siRNA) and HeLa cell extracts. This protocol provides an Ago loading assay to study RNAi programming when starting with crude or partially purified cell extracts. The Ago loading assay should prove useful for studying other Ago proteins using a variety of mammalian cell extracts.
authors.affiliation: Department of Biochemistry and Molecular Biology, School of Medicine, Southern Illinois University, Carbondale, IL, 62901, USA. ktgagnon@siu.edu.|Department of Chemistry and Biochemistry, Southern Illinois University, Carbondale, IL, 62901, USA. ktgagnon@siu.edu.
authors.forename: Keith T
authors.initials: KT
authors.lastname: Gagnon
mesh_terms: D060565:Argonaute Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D008958:Models, Molecular; D009690:Nucleic Acid Conformation; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism
chemical_list: C466422:AGO2 protein, human; D060565:Argonaute Proteins; D034741:RNA, Small Interfering
keywords: Ago2; Argonaute loading; Cell extract; Human; In vitro; Mammalian; RNA interference; RNAi programming; Small duplex RNA