scholarly journals Microfluidic characterization of macromolecular liquid–liquid phase separation

Lab on a Chip ◽  
2020 ◽  
Vol 20 (22) ◽  
pp. 4225-4234
Author(s):  
Anne Bremer ◽  
Tanja Mittag ◽  
Michael Heymann
Keyword(s):  
Phase Separation ◽  
Liquid Phase ◽  
Precise Determination ◽  
Phase Morphology ◽  
Liquid Phase Separation ◽  
Dense Phase ◽  
Liquid Liquid Phase Separation

The microfluidic phase chip allows precise determination of the saturation concentrations of biomolecules that undergo liquid–liquid phase separation while also monitoring the dense-phase morphology.

scholarly journals Microfluidic characterization of macromolecular liquid-liquid phase separation

2020 ◽  
Author(s):  
Anne Bremer ◽  
Tanja Mittag ◽  
Michael Heymann
Keyword(s):  
Phase Separation ◽  
Liquid Phase ◽  
Liquid Phase Separation ◽  
Dense Phase ◽  
Low Protein ◽  
Quantitative Mapping ◽  
Reported Data ◽  
Liquid Liquid Phase Separation

AbstractLiquid-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 phase. We further show that the saturation concentrations measured in chip 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.

scholarly journals Capillary flow experiments for thermodynamic and kinetic characterization of protein liquid-liquid phase separation

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Emil G. P. Stender ◽  
Soumik Ray ◽  
Rasmus K. Norrild ◽  
Jacob Aunstrup Larsen ◽  
Daniel Petersen ◽  
...  
Keyword(s):  
Phase Separation ◽  
Liquid Phase ◽  
Amyloid Fibrils ◽  
Capillary Flow ◽  
Solid Phase ◽  
Liquid Phase Separation ◽  
Dead Box ◽  
Phase Concentration ◽  
Liquid Liquid Phase Separation

AbstractLiquid-liquid phase separation or LLPS of proteins is a field of mounting importance and the value of quantitative kinetic and thermodynamic characterization of LLPS is increasingly recognized. We present a method, Capflex, which allows rapid and accurate quantification of key parameters for LLPS: Dilute phase concentration, relative droplet size distributions, and the kinetics of droplet formation and maturation into amyloid fibrils. The binding affinity between the polypeptide undergoing LLPS and LLPS-modulating compounds can also be determined. We apply Capflex to characterize the LLPS of Human DEAD-box helicase-4 and the coacervate system ssDNA/RP3. Furthermore, we study LLPS and the aberrant liquid-to-solid phase transition of α-synuclein. We quantitatively measure the decrease in dilute phase concentration as the LLPS of α-synuclein is followed by the formation of Thioflavin-T positive amyloid aggregates. The high information content, throughput and the versatility of Capflex makes it a valuable tool for characterizing biomolecular LLPS.

scholarly journals Prediction and characterization of liquid-liquid phase separation of minimalistic peptides

2021 ◽  
pp. 100579
Author(s):  
Yiming Tang ◽  
Santu Bera ◽  
Yifei Yao ◽  
Jiyuan Zeng ◽  
Zenghui Lao ◽  
...  
Keyword(s):  
Phase Separation ◽  
Liquid Phase ◽  
Liquid Phase Separation ◽  
Liquid Liquid Phase Separation

scholarly journals A generic approach to study the kinetics of liquid–liquid phase separation under near-native conditions

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Joris Van Lindt ◽  
Anna Bratek-Skicki ◽  
Phuong N. Nguyen ◽  
Donya Pakravan ◽  
Luis F. Durán-Armenta ◽  
...  
Keyword(s):  
Phase Separation ◽  
Liquid Phase ◽  
Cell Biology ◽  
Molecular Mechanisms ◽  
Stress Protein ◽  
Low Complexity ◽  
Liquid Phase Separation ◽  
Phase Separation Behavior ◽  
Liquid Liquid Phase Separation

AbstractUnderstanding the kinetics, thermodynamics, and molecular mechanisms of liquid–liquid phase separation (LLPS) is of paramount importance in cell biology, requiring reproducible methods for studying often severely aggregation-prone proteins. Frequently applied approaches for inducing LLPS, such as dilution of the protein from an urea-containing solution or cleavage of its fused solubility tag, often lead to very different kinetic behaviors. Here we demonstrate that at carefully selected pH values proteins such as the low-complexity domain of hnRNPA2, TDP-43, and NUP98, or the stress protein ERD14, can be kept in solution and their LLPS can then be induced by a jump to native pH. This approach represents a generic method for studying the full kinetic trajectory of LLPS under near native conditions that can be easily controlled, providing a platform for the characterization of physiologically relevant phase-separation behavior of diverse proteins.

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