Argonaute Proteins, 2011 Methods and Protocols Methods in Molecular Biology Series, Vol. 725

With the rapid proliferation of RNAi applications in basic and clinical sciences, the challenge has now become understanding how components of RNAi machinery function together in a regulated manner.  Argonaute proteins are the central effectors of RNAi and are highly conserved among eukaryotes and some archaebacteria. These RNA-binding proteins use small guide RNAs to silence expression of genes at the mRNA and DNA levels.  In Argonaute Proteins: Methods and Protocols, expert researchers in this burgeoning field provide detailed, up-to-date methods to study Argonaute protein functions and interactions in a wide variety of cell types ranging from yeast to mammalian systems, as well as in vitro.  Written in the highly successful Methods in Molecular Biology? series format, chapters include brief introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and key tips on troubleshooting and avoiding known pitfalls.

1. Purification of Native Argonaute Complexes from the Fission Yeast Schizosaccharomycespombe

            Shane M. Buker and Mohammad R. Motamedi

2. Chromatin Immuno-Precipitation in Fission Yeast

            Thomas A. Volpe and Jessica DeMaio

3. Biochemical Analyses of Endogenous Argonaute Complexes Immunopurified with Anti-Argonaute Monoclonal Antibodies

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

4. Mapping of Ago2-GW182 Functional Interactions

            Bing Yao, Songqing Li, Shangli Lian, Marvin J. Fritzler, and Edward K.L. Chan

5. Continuous Density Gradients to Study Argonaute and GW182 Complexes Associated with the Endocytic Pathway

            Derrick Gibbings

6. In vitro RISC Cleavage Assay

            Julia Stoehr and Gunter Meister

7. Native Gel Analysis for RISC Assembly

            Tomoko Kawamata and Yukihide Tomari

8. Purification and Assembly of Human Argonaute, Dicer, and TRBP Complexes

            Nabanita De and Ian J. MacRae

9. Detection of Human Dicer and Argonaute 2 Catalytic Activity

            Marjorie P. Perron, Patricia Landry, Isabelle Plante, and Patrick Provost

10. Imaging the Cellular Dynamics of Drosophila Argonaute Proteins

            Jing Li, Nima Najand, Wendy Long, and Andrew Simmonds

11. ­­­­Live Cell Imaging of Argonaute Proteins in Mammalian Cells

            Justin M. Pare, Joaquin Lopez-Orozco, and Tom C. Hobman

12. Reporter-Based Assays for Analyzing RNA Interference in Mammalian Cells

            Lydia V. McClure, Gil Ju Seo, and Christopher S. Sullivan

13. Artificial Tethering of Argonaute Proteins for Studying their Role in Translational Repression of Target mRNAs

            Stephanie Eckhardt, Emilia Szostak, Zhaolin Yang, and Ramesh Pillai

14. An Efficient System For Let-7 MicroRNA- And GW182 Protein-Mediated Deadenylation In vitro

            Marc R. Fabian, Yuri V. Svitkin, and Nahum Sonenberg

15. Cell-Free microRNA-Mediated Translation Repression in C. elegans

            Edlyn Wu and Thomas F. Duchaine

16. Argonaute Pull-Down and RISC Analysis Using 2'-O-Methylated Oligonucleotides Affinity Matrices

            Guillaume Jannot, Alejandro Vasquez-Rifo, and Martin J. Simard

17. Cloning Argonaute-Associated Small RNAs from C. elegans

            Weifeng Gu, Julie M. Claycomb, Pedro J Batista, Craig C. Mello, and Darryl Conte, Jr.

18. Immunoprecipitation of piRNPs and Directional, Next Generation Sequencing of piRNAs

            Yohei Kirino, Anastassios Vourekas, Eugene Khandros, and Zissimos Mourelatos

19. Generation of an Inducible Mouse ES Cell Line Deficient for Argonaute Proteins

            Hong Su and Xiaozhong Wang

20. Whole Cell Proteome Regulation by microRNAs Captured in a Pulsed SILAC Mass Spectrometry Approach