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Biochemical isolation of Argonaute protein complexes by Ago-APP.

ABSTRACT: During microRNA (miRNA)-guided gene silencing, Argonaute (Ago) proteins interact with a member of the TNRC6/GW protein family. Here we used a short GW protein-derived peptide fused to GST and demonstrate that it binds to Ago proteins with high affinity. This allows for the simultaneous isolation of all Ago protein complexes expressed in diverse species to identify associated proteins, small RNAs, or target mRNAs. We refer to our method as "Ago protein Affinity Purification by Peptides" (Ago-APP). Furthermore, expression of this peptide competes for endogenous TNRC6 proteins, leading to global inhibition of miRNA function in mammalian cells.

SUBMITTER: Hauptmann J

PROVIDER: S-EPMC4586862 | biostudies-literature | 2015 Sep

REPOSITORIES: biostudies-literature

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Biochemical isolation of Argonaute protein complexes by Ago-APP.

Hauptmann Judith J Schraivogel Daniel D Bruckmann Astrid A Manickavel Sudhir S Jakob Leonhard L Eichner Norbert N Pfaff Janina J Urban Marc M Sprunck Stefanie S Hafner Markus M Tuschl Thomas T Deutzmann Rainer R Meister Gunter G

Proceedings of the National Academy of Sciences of the United States of America 20150908 38

During microRNA (miRNA)-guided gene silencing, Argonaute (Ago) proteins interact with a member of the TNRC6/GW protein family. Here we used a short GW protein-derived peptide fused to GST and demonstrate that it binds to Ago proteins with high affinity. This allows for the simultaneous isolation of all Ago protein complexes expressed in diverse species to identify associated proteins, small RNAs, or target mRNAs. We refer to our method as "Ago protein Affinity Purification by Peptides" (Ago-APP) ...[more]

PMID: 26351695

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