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Capillary Flow Experiments (Capflex) for Thermodynamic and Kinetic Characterization of Protein LLPS at High Throughput

preprint
submitted on 22.03.2021, 21:13 and posted on 23.03.2021, 13:08 by Emil G. P. Stender, Rasmus Krogh Norrild, Jacob Aunstrup Larsen, Henrik Jensen, Alexander Buell
Liquid-liquid phase separation (LLPS) of proteins is a field of mounting importance to life sciences. We present a new method, Capflex, that can easily be automated, allowing rapid and accurate quantification of key parameters for LLPS. Dilute phase concentration, relative droplet size distributions and the kinetics of droplet formation are quantified. Uniquely, the binding affinity between the polypeptide undergoing LLPS and LLPS-modulating compounds can also be determined. We applied Capflex to characterize the LLPS of Ddx4n1 and found that PEG3000 and Ca2+ promotes LLPS while ssDNA is detrimental. Furthermore, we characterized the membraneless organelle model system RP3 and provide the first experimentally recorded affinity of RP3 for DNA. We believe the high information content and high throughput of Capflex makes it a valuable tool for characterizing biomolecular LLPS.

Funding

NNFSA170028392

CF19-0055

NNFOC0055625

History

Email Address of Submitting Author

alebu@dtu.dk

Institution

Technical University of Denmark

Country

Denmark

ORCID For Submitting Author

0000-0003-1161-3622

Declaration of Conflict of Interest

Henrik Jensen is the CSO of FIDA Biosystems, the manufacturer of the instrument used in this study. However, the authors declare no conflict of interest.

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