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This volume provides readers with a broad collection of theoretical, computational, and experimental methods to quantitatively study the properties of phase-separate biomolecular condensates in diverse systems. The chapters in this book cover topics such as theoretical and computational methods; methods for in vitro characterization of biomolecular condensates; and techniques that enable in-cell characterization of biomolecular condensates. Written in the highly successful Methods in Molecular Biology series format, chapters include introduction to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and expert tips on troubleshooting and avoiding known pitfalls.
Comprehensive and thorough, Phase-Separated Biomolecular Condensates: Methods and Protocols is a valuable resource that helps researchers learn and use established methods to study both biophysical properties and biological functions of biomolecular condensates.
List of contents
Calculating Binodals and Interfacial Tension of Phase-Separated Condensates from Molecular Simulations, with Finite-Size Corrections.- Field-Theoretic Simulation Method to Study the Liquid-Liquid Phase Separation of Polymers.- Numerical Techniques for Applications of Analytical Theories to Sequence-Dependent Phase Separations of Intrinsically Disordered Proteins.- An Introduction to the Stickers-and-Spacers Framework as Applied to Biomolecular Condensates.- Multi-Scale Modeling of Protein-RNA Condensation In and Out of Equilibrium.- Fluorescence Lifetime Imaging Microscopy of Biomolecular Condensates.- Single-Molecule Fluorescence Methods to Study Protein-RNA Interactions Underlying Biomolecular Condensates.- Fluorescence Correlation Spectroscopy and Phase Separation.- Measurement of Protein and Nucleic Acid Diffusion Coefficients within Biomolecular Condensates Using In-Droplet Fluorescence Correlation Spectroscopy.- Single-Molecule Imaging of the Phase Separation-Modulated DNA Compaction to Study Transcriptional Repression.- Phase Separation-Based Biochemical Assays for Biomolecular Interactions.- Determining Thermodynamic and Material Properties of Biomolecular Condensates by Confocal Microscopy and Optical Tweezers.- A High-Throughput Method to Profile Protein Liquid-Liquid Phase Separation.- Phase Separation of Rubisco by the Folded SSUL Domains of CcmM in Beta-Carboxysome Biogenesis.- Cryo-Electron Tomography of Reconstituted Biomolecular Condensate.- Sedimentation Assays to Assess the Impact of Post-Translational Modifications on Phase Separation of RNA-Binding Proteins In Vitro and In Cells.- Synthetic Organelles for Multiple mRNA Selective Genetic Code Expansion in Eukaryotes.- Single Molecule Tracking of RNA Polymerase In and Out of Condensates in Live Bacterial Cells.- An Optogenetic Toolkit for the Control of Phase Separation in Living Cells.- Assessing the Phase Separation Propensity of Proteins in Living Cells Through Optodroplet Formation.- Mass Balance Imaging: A Phase Portrait Analysis for Characterizing Growth Kinetics of Biomolecular Condensates.- Characterizing Properties of Biomolecular Condensates Below the Diffraction Limit In Vivo.
