Related Experiment Video
Updated: Jun 2, 2026

Single-step Purification of Macromolecular Complexes Using RNA Attached to Biotin and a Photo-cleavable Linker
Published on: January 3, 2019
Purification and assembly of human Argonaute, Dicer, and TRBP complexes
1Department of Molecular Biology, The Scripps Research Institute, La Jolla, CA, USA.
Abstract:
The RNA-induced silencing complex (RISC) is a programmable gene-silencing machine involved in many aspects of eukaryotic biology. In humans, RISC is programmed or "loaded" with a small-guide RNA by the action of a tri-molecular assembly termed the RISC-loading complex (RLC). The human RLC is composed of the proteins Dicer, TRBP, and Argonaute2 (Ago2). To facilitate structural and biochemical dissection of the RISC-loading process, we have developed a system for the in vitro reconstitution of the human RLC. Here, we describe in detail methods for the expression and purification of recombinant Dicer, TRBP, and Ago2 and protocols for the assembly of RLCs and RLC subcomplexes. We also describe several simple assays to observe the biochemical activities of the assembled protein complexes.
Related Concept Videos
Protein Complex Assembly
Many viruses self-assemble into a fully functional unit using the infected host cell to...
Protein Complex Assembly
Many viruses self-assemble into a fully functional unit using the infected host cell to...
piRNA - Piwi-interacting RNAs
Conservation of Protein Domains Over Different Proteins
A limited set of protein domains often duplicate and recombine during evolution. These domains can be organized in different combinations to form...
Ribosomal RNA Synthesis
Ribosome biogenesis begins with the synthesis of 5S and 45S pre-rRNAs by distinct RNA polymerases. The primary transcripts are extensively processed and modified before they are bound and folded by ribosomal proteins and assembly factors,...
Coat Assembly and GTPases
Coat assembly depends on the local availability of phosphatidylinositol phosphates or PIPs and GTP-binding proteins. Adaptor proteins, which link the coat proteins to the membrane, bind to these PIPs and play a crucial role in controlling...

