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  • High-fidelity reconstitution of stress granules and nucleoli in mammalian cellular lysate.

High-fidelity reconstitution of stress granules and nucleoli in mammalian cellular lysate.

The Journal of cell biology (2021-01-28)
Brian D Freibaum, James Messing, Peiguo Yang, Hong Joo Kim, J Paul Taylor
ABSTRACT

Liquid-liquid phase separation (LLPS) is a mechanism of intracellular organization that underlies the assembly of a variety of RNP granules. Fundamental biophysical principles governing LLPS during granule assembly have been revealed by simple in vitro systems, but these systems have limitations when studying the biology of complex, multicomponent RNP granules. Visualization of RNP granules in cells has validated key principles revealed by simple in vitro systems, but this approach presents difficulties for interrogating biophysical features of RNP granules and provides limited ability to manipulate protein, nucleic acid, or small molecule concentrations. Here, we introduce a system that builds upon recent insights into the mechanisms underlying RNP granule assembly and permits high-fidelity reconstitution of stress granules and the granular component of nucleoli in mammalian cellular lysate. This system fills the gap between simple in vitro systems and live cells and allows for a variety of studies of membraneless organelles, including the development of therapeutics that modify properties of specific condensates.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
1,6-Hexanediol, 97%
Sigma-Aldrich
Anti-B23 antibody, Mouse monoclonal, clone FC82291, purified from hybridoma cell culture
Sigma-Aldrich
TEV Protease
Sigma-Aldrich
Cycloheximide, from microbial, ≥94% (TLC)
Grace Bio-Labs SecureSeal imaging spacer, 1 well, diam. × thickness 9 mm × 0.12 mm