Argonaute Proteins

Part of the respected Methods in Molecular Biology(TM) series, this practical volume features brief introductions to the key topics, lists the necessary materials and reagents, and includes reproducible laboratory protocols as well as key troubleshooting tips.

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.

Practical and authoritative, Argonaute Proteins: Methods and Protocols serves as a vital reference for both experienced and novice scientists approaching the vast complexities of RNAi research.

Aids researchers in examining these versatile proteins in a wide variety of cell types Features practical, step-by-step protocols which open the door to future progress in the field of molecular therapeutics Contains expert tips and vital implementation advice to ensure success

Inhalt

Purification of Native Argonaute Complexes from the Fission Yeast Schizosaccharomyces pombe.- Chromatin Immuno-Precipitation in Fission Yeast.- Biochemical Analyses of Endogenous Argonaute Complexes Immunopurified with Anti-Argonaute Monoclonal Antibodies.- Mapping of Ago2-GW182 Functional Interactions.- Continuous Density Gradients to Study Argonaute and GW182 Complexes Associated with the Endocytic Pathway.- In vitro RISC Cleavage Assay.- Native Gel Analysis for RISC Assembly.- Purification and Assembly of Human Argonaute, Dicer, and TRBP Complexes.- Detection of Human Dicer and Argonaute 2 Catalytic Activity.- Imaging the Cellular Dynamics of Drosophila Argonaute Proteins.- Live Cell Imaging of Argonaute Proteins in Mammalian Cells.- Reporter-Based Assays for Analyzing RNA Interference in Mammalian Cells.- Artificial Tethering of Argonaute Proteins for Studying their Role in Translational Repression of Target mRNAs.- An Efficient System For Let-7 MicroRNA- And GW182 Protein-Mediated Deadenylation In vitro.- Cell-Free microRNA-Mediated Translation Repression in C. elegans.- Argonaute Pull-Down and RISC Analysis Using 2'- O -Methylated Oligonucleotides Affinity Matrices.- Cloning Argonaute-Associated Small RNAs from C. elegans.- Immunoprecipitation of piRNPs and Directional, Next Generation Sequencing of piRNAs.- Generation of an Inducible Mouse ES Cell Line Deficient for Argonaute Proteins.- Whole Cell Proteome Regulation by microRNAs Captured in a Pulsed SILAC Mass Spectrometry Approach.