Argonaute Proteins

Methods and Protocols
2011
ISBN: 978-1-61779-045-4
Verlag: Humana Press

Buch, Englisch, Band 725, 344 Seiten, Format (B × H): 183 mm x 260 mm, Gewicht: 867 g

Reihe: Methods in Molecular Biology

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.

Zielgruppe

Professional/practitioner

Autoren/Hrsg.

Duchaine, Thomas F.

Hobman, Tom C.

Fachgebiete

• Naturwissenschaften Biowissenschaften Biowissenschaften Genetik und Genomik (nichtmedizinisch)
• Technische Wissenschaften Verfahrenstechnik | Chemieingenieurwesen | Biotechnologie Biotechnologie
• Naturwissenschaften Biowissenschaften Proteinforschung
• Naturwissenschaften Biowissenschaften Molekularbiologie

Inhaltsverzeichnis

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