Detection and Analysis of Liquid-Liquid Phase Separation

Liquid-liquid phase separation (LLPS) of proteins, RNA or DNA can lead to the formation of various biomolecular condensates, and LLPS represents a common mechanical principle of intracellular spatial organization. As an ideal partner for biomolecular condensates, CD Biosciences provides quantitative tools or methods to help customers worldwide detect and analyze liquid-liquid phase separation.

The Importance of LLPS

LLPS is a thermodynamically driven process in which a cellular environment with a relatively well-mixed distribution of biomolecules is broken down into liquid droplets. LLPS of biomolecules has become a cornerstone mechanism for understanding the principles of intracellular compartmentalization, membraneless organelle (MLO) formation, and gene regulation. This understanding has led researchers to make significant efforts to characterize the function of such biomolecular condensates in normal cells and their role in contexts ranging from development to age-related diseases. To gain insight into the fundamental biophysical principles and specific properties of biomolecular condensates, clear criteria are needed for LLPS research to analyze a wide range of biological processes and to elucidate the causal relationship between abnormal LLPS and the development of pathological conditions.

Fig. 1. Overview of experimental approaches used to evaluate properties of assemblies formed by LLPS. (Alberti S, et al, 2019)

Customized Services

Our team of experts is interested in understanding the fundamental biophysical principles and specific properties of biological condensates, and we are committed to helping our clients gain insight into a wide range of biological processes and systems. CD BioSciences develops clear research criteria to analyze the surge in the physiological and pathological context of LLPS. We hope that the proposed protocol will enable our clients to perform protein LLPS experiments easily and efficiently.

Here, detailed, fully automated, high-throughput analysis of LLPS is fully feasible. We offer cutting-edge methods for determining dilute phase concentration, relative droplet size distribution, kinetics of droplet formation, maturation of concentrated proteins into amyloidogenic fibrils, and binding affinity between LLPS-passed peptides and LLPS-modulated compounds, etc. CD BioSciences offers a comprehensive range of methods for identifying and studying liquid-liquid phase separations.

• Visualization-Based Characterization of Liquid-Liquid Phase Separation
  We have advanced modern microscopy platforms to visualize biomolecular condensates, accelerating our customers' in vivo and ex vivo phase separation studies of biomolecular condensates. Our various microscopy techniques can provide detailed information about biomolecular condensates, including differential interference contrast (DIC), electron microscopy, confocal microscopy and super-resolution imaging techniques, etc.
• Stream Methods Based Characterization of Liquid-Liquid Phase Separation
  We offer advanced stream-based methods to analyze the complete kinetic trajectory of LLPS under easily controlled near-natural conditions, which are applicable to any aggregation-prone protein.
• Scattering Methods Based Characterization of Liquid-Liquid Phase Separation
  We offer scattering methods of electromagnetic radiation at different wavelengths for characterizing LLPS, including determining protein structure, characterizing correlation distances within the phase separation, and quantifying changes in protein and nucleic acid chain compression, etc.
• Nuclear Magnetic Resonance Based Characterization of Liquid-Liquid Phase Separation
  We provide nuclear magnetic resonance (NMR) spectroscopy to analyze the conformational changes of intrinsically disordered proteins (IDPs) upon phase separation. Among them, the chemical shifts of ¹H, ¹³C, ¹⁵N, ¹⁹F and ³¹P nuclei make it possible to study the dynamic changes of the main chain and side chains of proteins during LLPS.
• Cryo-Electron Tomography Based Characterization of Liquid-Liquid Phase Separation
  We offer cryo-electron tomography as an ideal tool to characterize LLPS, allowing the study of the three-dimensional structure of condensates of this polymorphic interaction network at nanometer resolution, leading to inferences about function.
• Prediction of Liquid-Liquid Phase Separation
  Most proteins are capable of LLPS, but not all proteins can be subjected to LLPS under physiological conditions. We have several databases of proteins that undergo liquid-liquid phase separation and contain experimentally obtained data. In addition, we develop phase separation predictors to identify common features shared by all LLPS proteins from the available data.
• In Vitro Liquid-Liquid Phase Separation Assays
  LLPS, driven by multivalent macromolecular interactions, is an important organizing principle for biomolecular condensates. We offer nephelometric experiments, which probe LLPS by measuring the optical density at a fixed wavelength (350 nm or 600 nm). In addition, we have various cutting-edge microscopy platforms to observe droplets
• Induction of Liquid-Liquid Phase Separation In Vivo
  We offer several strategies to drive intracellular LLPS, including overexpression of scaffold proteins, promotion of their oligomerization, formation/disruption of complexes with partner proteins, increased intracellular crowding (e.g., osmotic stress), and the use of some specific stimuli.

CD BioSciences has a wealth of selected bioinformatics tools or databases for studying LLPS, and cutting-edge microscopy platforms to provide detailed information about LLPS. Each project will be individually designed to meet the client's requirements and described in a detailed project report. If you are interested in our services, please contact us.