Construction of Biomolecular Condensates Phase Diagrams

The liquid-liquid phase separation (LLPS) process is the fundamental mechanism describing the formation of biological condensates in living cells. Many cellular protein and nucleic acid molecules are capable of layering into a dense liquid condensed phase and a dilute biomolecule-depleted phase. Intracellular protein phase separation is sensitive to a variety of physicochemical parameters, including protein concentration, temperature, ionic strength, salt type, pH, presence of co-solutes, etc. The central concept in the characterization of phase separation systems is the phase diagram. A typical phase diagram can be constructed by varying protein concentration and temperature (or pH, salt concentration, etc.). A set of conditions must be defined so that the macromolecules in question form a single, well-mixed phase, and a set of conditions so that their phases separate, to generate the phase diagram.

Fig. 1. Schematic representation of a phase diagram. ( Alberti S, et al., 2019)

Customized Services

The phase diagrams of biomolecular condensates depend on a large number of molecular modifiers, environmental conditions, and other specific molecules that directly interact with the phase separation components. Here, CD BioSciences offers professional services to construct phase diagrams of biomolecular condensates that can quantify the physical parameters that modulate phase separation behaviour with high fidelity, accuracy and throughput in LLPS systems.

• Classical Phase Diagram Construction

(1) Systematically vary two conditions (e.g., protein and salt concentration) while holding other conditions constant (e.g., temperature and pH), and observe the conditions under which a dense phase is formed.

(2) Identify a region that delineates the dense and light phases by constructing a phase diagram, and indicate the conditions under which the system forms the two phases by tying lines connecting the two phases within that region.

✓ A large number of samples are required.

✓ Gradual combination of reagents.

✓ Explores a limited set of conditions and/or provides a coarse-grained diagram of protein phase space when performed manually.

✓ Allows analysis of the effect of physicochemical properties and valence of macromolecules on phase separation behaviour.

• Droplet-Based Microfluidics for Phase Diagram of Biomolecular Condensates

Due to the wide variety of proteins that undergo LLPS and the environmental conditions that regulate their behavior, we offer experimental methods for rapid and high-resolution characterization of LLPS phase diagrams. CD BioSciences is developing a state-of-the-art microfluidic platform for rapid and high-resolution acquisition of protein phase diagrams in multiple chemical and physical dimensions. The advantages of this approach are:

✓ Can characterize the phase behavior of various protein systems under various conditions.

✓ Can be used to quantitatively characterize the effect of small molecules on the phase transition of biomolecular condensates.

✓ Reduces the amount of sample needed to evaluate the phase diagram.

Phase diagrams of biomolecular condensates are used to investigate the phase behaviour of the condensate by determining the location of phase boundaries in chemical space and by monitoring the location of transitions between phase separation and mixing states. Our experts can construct phase diagrams based on a large number of individual measurements that sample different parts of the parameter space. 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.