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Microfluidic characterization of macromolecular liquid-liquid phase separation.

ABSTRACT: Liquid-liquid phase separation plays important roles in the compartmentalization of cells. Developing an understanding of how phase separation is encoded in biomacromolecules requires quantitative mapping of their phase behavior. Given that such experiments require large quantities of the biomolecule of interest, these efforts have been lagging behind the recent breadth of biological insights. Herein, we present a microfluidic phase chip that enables the measurement of saturation concentrations over at least three orders of magnitude for a broad spectrum of biomolecules and solution conditions. The phase chip consists of five units, each made of twenty individual sample chambers to allow the measurement of five sample conditions simultaneously. The analytes are slowly concentrated via evaporation of water, which is replaced by oil, until the sample undergoes phase separation into a dilute and dense phase. We show that the phase chip lowers the required sample quantity by 98% while offering six-fold better statistics in comparison to standard manual experiments that involve centrifugal separation of dilute and dense phases. We further show that the saturation concentrations measured in chips are in agreement with previously reported data for a variety of biomolecules. Concomitantly, time-dependent changes of the dense phase morphology and potential off-pathway processes, including aggregation, can be monitored microscopically. In summary, the phase chip is suited to exploring sequence-to-binodal relationships by enabling the determination of a large number of saturation concentrations at low protein cost.

SUBMITTER: Bremer A

PROVIDER: S-EPMC7658026 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Three archetypical classes of macromolecular regulators of protein liquid-liquid phase separation.

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2021-12-23 | GSE174575 | GEO

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| S-EPMC8674230 | biostudies-literature

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Project description:Bacterial Heterochromatin Formation Through Liquid-Liquid Phase Separation

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2022-10-20 | PXD033048 | Pride

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