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Protocol for the determination of intracellular phase separation thresholds.
Anna K Lee1, Patrick Ryan Potts1
1Department of Cell and Molecular Biology, St. Jude Children's Research Hospital, Memphis, TN 38105, USA.
STAR Protocols
|February 8, 2021
Summary
This study introduces a new method to measure protein phase separation within cells. It uses fluorescently tagged proteins to determine concentration thresholds, overcoming limitations of previous in vitro studies.
Area of Science:
- Biochemistry
- Cell Biology
- Molecular Biology
Background:
- Phase separation is crucial for cellular organization but is difficult to study in native conditions.
- Previous studies relied on in vitro assays with non-native conditions and hard-to-purify proteins.
Purpose of the Study:
- To establish a protocol for determining protein concentration thresholds for phase separation in living cells.
- To provide a more physiologically relevant method for studying phase separation.
Main Methods:
- Utilizes fluorescent imaging of Green Fluorescent Protein (GFP)-tagged proteins within cells.
- Determines relative protein concentration thresholds that induce phase separation.
Main Results:
- Successfully demonstrates a method for quantifying phase separation in situ.
- Establishes a protocol adaptable for various phase-separating proteins.
Conclusions:
- This in-cell protocol offers a powerful alternative to in vitro methods for studying protein phase separation.
- Advances in gene editing and reagent availability facilitate broad application of this technique.
