Measuring liquid-liquid phase separation with FIDA: a conversation with Prof. Alexander Buell

Prof. Alexander Buell is a professor of protein biophysics at the Technical University of Denmark (DTU), where he runs a research group and a protein-biophysics core facility working on protein aggregation, amyloid fibril formation, liquid-liquid phase separation (LLPS), protein materials and food proteins. In a conversation with Fidabio, Prof. Buell described how Flow Induced Dispersion Analysis (FIDA) fits that work. This is a summary of what he said.

Why Prof. Buell chose FIDA for LLPS

In liquid-liquid phase separation, a protein solution spontaneously separates into dense droplets and a much more dilute phase. Two questions dominate the biophysics: quantifying the thermodynamic driving forces behind it, and understanding how compounds enhance or inhibit it. Both demand methods that measure at high throughput on very little sample. Powerful microdroplet and microfluidic approaches exist, but they can be very difficult to use, whereas FIDA can be picked up quickly.

"Having a method available that allows you to measure at high throughput, but at the same time is based on a commercially available platform that doesn't need years of training, is really a valuable addition," Prof. Buell said.

What FIDA makes possible for phase-separation screening

The capability the Buell group relies on is quantifying phase-separation thermodynamics and screening many conditions from a tiny amount of material, which is exactly what testing many sequence variants, or screening candidate inhibitors, requires. In work published in Angewandte Chemie International Edition, the group induces phase separation inside the instrument itself: the protein is injected together with a component that represses phase separation, and as Taylor dispersion inside the capillary removes that component, phase separation is triggered, so the effect of other components can then be measured. The whole experiment uses only about 50 nanolitres of sample per data point on the Fida 1, and that low-sample, automated screening of tens or hundreds of conditions is what Prof. Buell singled out as unique.

"The capability of using just tens of nanolitres of sample and, in an automated way, screening tens or hundreds of conditions is quite unique to FIDA, and we don't have any other method in our lab that would be able to do that," Prof. Buell said.

How it sits alongside other techniques

FIDA is complementary, not a replacement. Light-scattering methods remain the more sensitive choice for tiny concentrations of aggregates or nanoclusters, and the thermodynamics can also be reached more simply, for instance by centrifuging a sample to separate its dense and dilute phases. FIDA's distinction is the combination of very little sample and automated, many-condition screening. The group also published a comprehensive review of the methods available to quantify phase-separation thermodynamics, placing its FIDA-based approach alongside the others.

Where the work is heading

Alongside routine binding-affinity measurements, the Buell group has moved to micellar species in the food-protein space. Casein in milk, for example, forms micelles of various sizes and complexes calcium, and FIDA has proved useful not only to quantify micelle formation but to follow the dynamics, how stable the micelles are as conditions change, such as on removal of calcium.

What we learned from this conversation

• Thermodynamics from tens of nanolitres: quantify phase-separation driving forces and screen tens to hundreds of conditions on a turnkey platform.
• Phase separation induced in the capillary: Taylor dispersion removes a repressor to trigger it, then the effect of added components is measured, at about 50 nanolitres per data point on the Fida 1.
• Low barrier to entry: commercially available and learnable in a short time, unlike specialist microdroplet methods.
• Complementary to light scattering; also extending to micellar systems such as casein.

Curious how FIDA measures size, binding and phase behaviour in solution? Explore the FIDA technology and the Fida instrument, or browse the peer-reviewed literature. Read more scientists’ experiences with FIDA.