The kinetics, thermodynamics, and molecular mechanisms of liquid-liquid phase separation (LLPS) are critical in cell biology. We are developing reproducible stream-based methods to analyze the LLPS kinetics of proteins that are often severely aggregation-prone. Here at CD BioSciences, we offer a powerful and easy-to-use stopped-flow experimental platform to enable such measurements.

Introduction

LLPS is involved in the formation of biomolecular cohesions. Assessing the biological significance of LLPS requires an understanding of the kinetics of its biogenesis. The early chemical and biochemical processes of LLPS occur too rapidly to be analyzed using standard laboratory equipment. Stop-flow instrumentation is a fast kinetic technique for tracking chemical reactions on millisecond to second time scales. Stop-flow is currently used for a wide range of academic and industrial applications, including drug binding processes and protein stability. In a stopped-flow experiment, two, three, or four sample solutions are rapidly mixed and injected into an observation cell. When flow is stopped, kinetics are recorded with a detector best suited to the solution chemistry and information of interest (e.g., particle size, fluorophore environment, chromophores).

Fig. 1. Kinetics of phase separation of hnRNPA2 LCD induced by three different approaches. (Van Lindt J, et al., 2021)

Customized Services

The LLPS kinetics information can help the client understand the mechanism of biomolecular coalescence formation and its biological function. Based on the cutting-edge stopped-flow apparatus, CD BioSciences offers the stopped-flow experiment to track such rapid processes as the early stages of LLPS on millisecond time scales. This apparatus allows automated preparation of samples for analysis using relatively small volumes of two, three or four solutions mixed at high speed.

Attracting customers, our stopped-flow experiments are applicable to a variety of methods for inducing LLPS, including ionic strength reduction, dilution of concentrated denaturant solutions, addition of crowding agents, mixing with congeners, desolvation labeling, pH jumping, etc.

In addition, our stopped-flow apparatus can be used with a variety of detectors, thus making it available for ongoing research and development of LLPS. We combine the stopped-flow apparatus with a temperature jump apparatus that allows temperature jumps of up to 60°C to be performed on sub-millisecond time scales. We use this device to thermally sense LLPS and analyze the thermal regulation of the process. LLPS reaction kinetics can be followed by appropriate experiments and incorporated into systematic assay techniques such as absorbance, fluorescence intensity, fluorescence anisotropy, 90 degree light scattering, and circular dichroism.

Advantages of the Stopped-Flow Experiment

• The ability to study rapid reactions in solution on time scales ranging from 1 millisecond to hundreds of seconds.
• A wide range of reactions can be studied, such as protein-protein interactions, ligand binding, electron transfer, protein folding, chemical or coordination reactions.
• Analysis of the resulting transient kinetics can determine reaction rates, complexity of reaction mechanisms, information on short-lived reaction intermediates, etc.
• Can be used to show the effect of parameters such as temperature, pH and reagent concentration on the kinetics of LLPS.

Stop-flow experiments are widely used to increase customers' understanding of fast kinetics and are particularly useful for chemists and biochemists. We offer a versatile and easily adaptable method to study the kinetic pathways of LLPS. If you are interested in our services, please do not hesitate to contact us for more information.