In Vitro Liquid-Liquid Phase Separation Assays

Liquid-liquid phase separation (LLPS) or condensation is the basis for the formation of biomolecular condensates. As membrane-free subcellular compartments, biomolecular condensates play a plethora of important functions in signaling and storage. Biomolecular condensates are mainly composed of proteins and RNA and are maintained by molecular interactions. Similarly, multivalent interactions between proteins and RNA contribute to LLPS-mediated RNP particle formation, such as stress particles and P-bodies. Currently, in vitro LLPS assays are necessary to investigate further understanding of how the assembly of phase-separated compartments is regulated in cells. However, there are a lack of uniform standards and protocols for these in vitro studies.

Fig. 1. Assays used for examining in vitro phase-separated droplets. (Peran I, et al., 2020)

Customized Services

Our experts are interested in the basic biophysical principles and specific properties of biomolecular condensates, and are committed to the rigorous experimental characterization of LLPS. Our goal is to provide an initial practical experimental approach to the study of LLPS. Here, CD BioSciences offers standardized procedures and generally accepted LLPS analytical practices for the detection of turbidity, microscopic studies and dilute phase concentrations (C_L) of biomolecular condensates formed by LLPS.

• Turbidity Assays of Liquid-Liquid Phase Separation
  We offer turbidity meters, nephelometers, or spectrophotometers to detect changes in solution turbidity to determine the phase boundary of protein and/or RNA systems.
• Microscopic Studies of Liquid-Liquid Phase Separation
  We have cutting-edge optical microscopes that can observe these solutions under conditions that do not promote LLPS and also under conditions that change to promote droplet formation.

• Morphological assays: Protein droplets formed by LLPS usually appear as spherical droplets under the microscope.
• Droplet fusion and fission assays: Droplets fuse and form larger droplets to minimize surface tension. In addition, larger droplets can undergo both fission and dropping to form smaller droplets.
• Wetting test: These droplets typically have the ability to wet the surface.

• Determination of C_L by Centrifugation
  Accurate measurement of light or dense phase concentrations C_L and C_D can reflect light and dense phase separation. We offer a sedimentation method to quantitatively examine protein phase separation. The proportion of total protein assigned to the phase separated droplets is determined by measuring the intensity of the bands in the supernatant and precipitation samples.

Our in vitro strategy has the inherent advantage that it is performed with purified components and can explicitly test which components are critical to the process. CD BioSciences offers a detailed methodological protocol for characterizing condensate formation in vitro via LLPS. If you have any special requirements for our services, please feel free to contact us. We are looking forward to working together with your attractive projects.