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Updated: Jun 12, 2025
On-Chip Octanol-Assisted Liposome Assembly for Bioengineering
Published on: March 17, 2023
Protocol for validating liquid-liquid phase separation as a driver of membraneless organelle assembly in vitro and in
Marius Hedtfeld1, Andrea Musacchio1
1Department of Mechanistic Cell Biology, Max Planck Institute of Molecular Physiology, Otto-Hahn-Straße 11, 44227 Dortmund, Germany; Centre for Medical Biotechnology, Faculty of Biology, University Duisburg-Essen, Essen, Germany.
Abstract:
Liquid-liquid phase separation (LLPS) of scaffold proteins has often been proposed to drive the biogenesis of membraneless cellular compartments. Here, we present a protocol to link in vitro LLPS propensity to localization in vivo. We describe steps for examining LLPS in vitro in the presence of crowding agents or cytomimetic media. We complement our in vitro studies with recombinant proteins with experiments of protein electroporation into mitotic HeLa cells. In addition, we discuss steps to assess protein localization and delivery levels. For complete details on the use and execution of this protocol, please refer to Hedtfeld et al.1.
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