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Liquid-Liquid Phase Coexistence and Membraneless Organelles

1st Edition - January 14, 2021

Editor: Christine D. Keating

Hardback ISBN:

9 7 8 - 0 - 1 2 - 8 2 1 1 5 9 - 5

eBook ISBN:

9 7 8 - 0 - 1 2 - 8 2 1 1 6 0 - 1

Methods in Enzymology, Volume 646, continues the legacy of this premier serial with quality chapters authored by leaders in the field. Chapters in this new release include Methods… Read more

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Methods in Enzymology, Volume 646, continues the legacy of this premier serial with quality chapters authored by leaders in the field. Chapters in this new release include Methods for Studying RNA condensation/granules in vitro, RNA Dynamics in Intracellular Condensates, Methods for Viscoelastic Characterization of Liquid and Gel Condensates, Incorporating Proteins into Complex Coacervates, Methods for Study of Liquid-Liquid Phase Coexistence in Proximity to Lipid Membranes, Preparation of and Solute Partitioning in Multiphase Coacervates, Reversible photocontrol of DNA coacervation, Enzymatic Control over Coacervation, and much more.

Cover image
Title page
Table of Contents
Copyright
Contributors
Preface
Chapter One: Using a sequence-specific coarse-grained model for studying protein liquid–liquid phase separation
Abstract
1: Introduction
2: HPS model
3: Simulation strategy for obtaining phase diagrams
4: Summary
Acknowledgments
Chapter Two: Reconstituting bacterial cell division assemblies in crowded, phase-separated media
Abstract
1: Introduction
2: Experimental approaches and analysis
3: Summary of experimental results
4: Concluding remarks and future prospects
Acknowledgments
Chapter Three: Terpolymer-stabilized complex coacervates: A robust and versatile synthetic cell platform
Abstract
1: Introduction
2: Synthesis of coacervate forming materials and terpolymer
3: Formation of terpolymer-stabilized coacervates
4: Purification strategies
5: Measurement considerations
6: Conclusions and future perspectives
Acknowledgments
Chapter Four: Cell-free reconstitution of multi-condensate assemblies
Abstract
1: Introduction
2: Considerations when working with BC proteins
3: Preparation of recombinant MEG-3 and PGL-3 proteins
4: Fluorescent labeling of protein and RNA
5: Condensation assays
6: Imaging and data analysis
7: Concluding remarks
Chapter Five: Practical considerations for generation of multi-compartment complex coacervates
Abstract
1: Introduction
2: Procedure
Acknowledgments
Chapter Six: Methods for characterizing the material properties of biomolecular condensates
Abstract
1: Introduction
2: Measuring mesoscale physical properties of liquid droplets using optical trap-induced droplet coalescence: Concepts
3: Measuring mesoscale physical properties of liquid droplets using optical trap-induced droplet coalescence: Methods
4: Determining nanoscale biomolecular diffusion within liquid droplets using fluorescence recovery after photobleaching (FRAP): Concepts
5: Determining diffusion dynamics within liquid droplets using fluorescence recovery after photobleaching: Methods
6: Single particle tracking (SPT)
7: Advanced methods
8: Conclusion
Acknowledgments
Chapter Seven: Small-angle X-ray scattering experiments of monodisperse intrinsically disordered protein samples close to the solubility limit
Abstract
1: Introduction
2: Resolution of structural information from SAXS
3: Complications of SAXS measurements of self-assembling proteins
4: IDR sample measurement in SEC-SAXS mode
5: Primary data analysis
6: Synchrotron SAXS beamline hardware
7: Model-dependent analysis
8: Implementation of SAXS measurements of single chain IDR behavior for characterizing phase behavior
9: Conclusions
Acknowledgments
Chapter Eight: SAXS methods for investigating macromolecular and self-assembled polyelectrolyte complexes
Abstract
1: Introduction
2: Macromolecular polyelectrolyte complexes (PECs)
3: Self-assembled PEC micelles
4: Self-assembled PEC hydrogels
5: Summary
Acknowledgments
Chapter Nine: Ultra-small angle neutron scattering to study droplet formation in polyelectrolyte complex coacervates
Abstract
1: Introduction
2: Model polyelectrolyte complex coacervate and liquid-liquid phase separation
3: USANS instrument and experimental practice
4: Related techniques
5: Safety considerations and standards
6: Pros and cons
7: Summary
Acknowledgments
Chapter Ten: Incorporation of proteins into complex coacervates
Abstract
1: Introduction
2: Materials, equipment, and reagents
3: Safety considerations
4: Protocols
5: Analysis and statistics
6: Summary
Acknowledgments
Chapter Eleven: Measuring the activity and structure of functional RNAs inside compartments formed by liquid-liquid phase separation
Abstract
1: Rationale
2: Preparation of reagents
3: RNA structure and function inside complex coacervates
4: Conclusion
Acknowledgements
Chapter Twelve: Reversible photocontrol of DNA coacervation
Abstract
1: Introduction
2: Photoswitchable coacervation: The azoTAB/dsDNA system
3: Synthesis and photophysical characterization of azoTAB
4: Photoswitchable DNA coacervation
5: Conclusions
Acknowledgments
Chapter Thirteen: Enzymatic control over coacervation
Abstract
1: Introduction
2: Enzymatic reactions in two phases
3: Control over coacervation by chain length and charge density
4: Protocols
5: Analysis
6: Summary and conclusion