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 capillary flow experimental platform to enable such measurements.

Introduction

LLPS is a fundamental mechanism for the formation of biomolecular cohesions, and has received significant attention in academic and industrial settings as a key process for life. However, physicochemical experiments to perform highly quantitative measurements of biomolecular LLPS are currently rare and often too laborious. In most cases, phase diagrams of LLPS are obtained only by centrifugation and manual measurement of dilute phase concentrations. A capillary-based instrument called "Flow Induced Dispersion Analysis (FIDA-1)" uses Taylor dispersion to analyze the parabolic hydrodynamic flow profile of liquids through capillaries. This technique is widely used to measure the apparent size of an object and the interaction of fluorescently labeled ligands with the analyte.

Fig. 1. Quantitative analysis of Ddx4n1 LLPS using Capflex. (Stender E G P, et al., 2021)

Customized Services

Based on the cutting-edge FIDA method, CD BioSciences offers the capillary experiments for detailed, fully automated, high-throughput analysis of LLPS. The method is widely used to study different aspects of LLPS, including:

✓ Droplet formation.

✓ Determination of the dilute phase concentration.

✓ Quantification of the relative droplet size distribution.

✓ Kinetics of droplet formation.

✓ Maturation of protein concentrates into amyloid protofibrils.

✓ Peptides after LLPS.

✓ Binding affinity between LLPS-modulated compounds.

We offer a customized procedure for capillary experiments to analyze LLPS. First, samples are started from a 96-well plate, maintained at a temperature above the cloud point, and LLPS is induced by pulse injection by placing the capillary in a chamber at a temperature below the cloud point. In addition, we also keep the solution composition of the sample and capillary filling buffer different to induce LLPS. Samples are then controllably carried over to a fluorescence detector where fluorescence time traces are recorded to analyze droplet size distribution and droplet formation kinetics.

Attractively for our customers, we use this method for the analysis of preformed droplets, to measure the affinity and complex size of droplet components in the non-phase separation region, and to study the effect of additives (e.g. Ca²⁺ and Mg²⁺ ions) and the molecular crowding agent on the driving force of biomolecular coalescence formation.

Advantages of the Capillary Flow Experiment

• A high-throughput, fully automated method.
• Up to 96 samples per day can be analyzed.
• Only a small amount of protein is required (20 mL per sample).
• Minimizes manual supervision.
• Suitable for biomolecular condensates with diameters of 0.5-1000 nm.

We offer a method that allows quantitative measurements of properties critical to LLPS, with complete phase diagrams. If you are interested in our services, please do not hesitate to contact us for more information.