For phase separation assays, polyanions were diluted to 1 µg/µL in 100 mM K2HPO4/KH2PO4 buffer at pH 7. Peptides were added at 250-µM concentrations. For partitioning experiments, probe molecules were spiked in at 100 nM. For the generation of pure RNA liquid droplets, homopolymeric RNAs were diluted to 2 µg/µL in 1x PBS buffer with 30% PEG and 10 mM MgCl2 at pH 7. For intradroplet FRAP, a circular area of 1-μM radius was bleached in droplets with a radius between 5 μM and 10 μM. For SXT 3D reconstructions, 92 projection images, with 200-ms exposure time each, were acquired sequentially around a rotation axis with 2° increment angles. For droplet fusion assays, confocal time-lapse images were taken of droplets settling on the coverslip and fusing together. Coarse-grained simulations are lattice-based and utilize a simple neighbor−neighbor interaction potential. For all other details, please see
Biomolecular Condensate Formation Assays
For phase separation assays, polyanions were diluted to 1 µg/µL in 100 mM K2HPO4/KH2PO4 buffer at pH 7. Peptides were added at 250-µM concentrations. For partitioning experiments, probe molecules were spiked in at 100 nM. For the generation of pure RNA liquid droplets, homopolymeric RNAs were diluted to 2 µg/µL in 1x PBS buffer with 30% PEG and 10 mM MgCl2 at pH 7. For intradroplet FRAP, a circular area of 1-μM radius was bleached in droplets with a radius between 5 μM and 10 μM. For SXT 3D reconstructions, 92 projection images, with 200-ms exposure time each, were acquired sequentially around a rotation axis with 2° increment angles. For droplet fusion assays, confocal time-lapse images were taken of droplets settling on the coverslip and fusing together. Coarse-grained simulations are lattice-based and utilize a simple neighbor−neighbor interaction potential. For all other details, please see
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Corresponding Organization :
Other organizations : Stanford University, Living Systems (United States), Lawrence Berkeley National Laboratory, University of California, San Francisco, Vlaams Instituut voor Biotechnologie, Vrije Universiteit Brussel, KU Leuven, HUN-REN Research Centre for Natural Sciences, Hungarian Academy of Sciences, Institute of Molecular Life Sciences
Protocol cited in 7 other protocols
Variable analysis
- Polyanion concentration (1 µg/µL)
- Peptide concentration (250 µM)
- Probe molecule concentration (100 nM)
- RNA concentration (2 µg/µL)
- PEG concentration (30%)
- MgCl2 concentration (10 mM)
- Phase separation
- Partitioning of probe molecules
- Formation of pure RNA liquid droplets
- Intradroplet FRAP measurements
- SXT 3D reconstructions
- Droplet fusion events
- Buffer composition (100 mM K2HPO4/KH2PO4 at pH 7 for phase separation; 1x PBS at pH 7 for RNA droplets)
- Droplet size (radius between 5 µM and 10 µM for intradroplet FRAP)
- Exposure time (200 ms per projection image for SXT)
- No positive or negative controls were explicitly mentioned in the input information.
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