Abstract
The formation of membrane-less organelles and compartments by protein phase separation is an important way in which cells organize their cytoplasm and nucleoplasm. In vitro phase separation assays with purified proteins have become the standard way to investigate proteins that form membrane-less compartments. By now, various proteins have been purified and tested for their ability to phase separate and form liquid condensates in vitro. However, phase-separating proteins are often aggregation-prone and difficult to purify and handle. As a consequence, the results from phase separation assays often differ between labs and are not easily reproduced. Thus, there is an urgent need for high-quality proteins, standardized procedures, and generally agreed-upon practices for protein purification and conducting phase separation assays. This paper provides protocols for protein purification and guides the user through the practicalities of in vitro protein phase separation assays, including best-practice approaches and pitfalls to avoid. We believe that this compendium of protocols and practices will provide a useful resource for scientists studying the phase behavior of proteins.
| Original language | English (US) |
|---|---|
| Pages (from-to) | 4806-4820 |
| Number of pages | 15 |
| Journal | Journal of Molecular Biology |
| Volume | 430 |
| Issue number | 23 |
| DOIs | |
| State | Published - Nov 2 2018 |
| Externally published | Yes |
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Keywords
- low-complexity proteins
- membrane-less compartment
- membrane-less organelle
- phase separation
- prion-like protein
- protein purification
- Sup35
ASJC Scopus subject areas
- Molecular Biology
Cite this
A User's Guide for Phase Separation Assays with Purified Proteins. / Alberti, Simon; Saha, Shambaditya; Woodruff, Jeffrey B.; Franzmann, Titus M.; Wang, Jie; Hyman, Anthony A.
In: Journal of Molecular Biology, Vol. 430, No. 23, 02.11.2018, p. 4806-4820.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - A User's Guide for Phase Separation Assays with Purified Proteins
AU - Alberti, Simon
AU - Saha, Shambaditya
AU - Woodruff, Jeffrey B.
AU - Franzmann, Titus M.
AU - Wang, Jie
AU - Hyman, Anthony A.
PY - 2018/11/2
Y1 - 2018/11/2
N2 - The formation of membrane-less organelles and compartments by protein phase separation is an important way in which cells organize their cytoplasm and nucleoplasm. In vitro phase separation assays with purified proteins have become the standard way to investigate proteins that form membrane-less compartments. By now, various proteins have been purified and tested for their ability to phase separate and form liquid condensates in vitro. However, phase-separating proteins are often aggregation-prone and difficult to purify and handle. As a consequence, the results from phase separation assays often differ between labs and are not easily reproduced. Thus, there is an urgent need for high-quality proteins, standardized procedures, and generally agreed-upon practices for protein purification and conducting phase separation assays. This paper provides protocols for protein purification and guides the user through the practicalities of in vitro protein phase separation assays, including best-practice approaches and pitfalls to avoid. We believe that this compendium of protocols and practices will provide a useful resource for scientists studying the phase behavior of proteins.
AB - The formation of membrane-less organelles and compartments by protein phase separation is an important way in which cells organize their cytoplasm and nucleoplasm. In vitro phase separation assays with purified proteins have become the standard way to investigate proteins that form membrane-less compartments. By now, various proteins have been purified and tested for their ability to phase separate and form liquid condensates in vitro. However, phase-separating proteins are often aggregation-prone and difficult to purify and handle. As a consequence, the results from phase separation assays often differ between labs and are not easily reproduced. Thus, there is an urgent need for high-quality proteins, standardized procedures, and generally agreed-upon practices for protein purification and conducting phase separation assays. This paper provides protocols for protein purification and guides the user through the practicalities of in vitro protein phase separation assays, including best-practice approaches and pitfalls to avoid. We believe that this compendium of protocols and practices will provide a useful resource for scientists studying the phase behavior of proteins.
KW - low-complexity proteins
KW - membrane-less compartment
KW - membrane-less organelle
KW - phase separation
KW - prion-like protein
KW - protein purification
KW - Sup35
UR - http://www.scopus.com/inward/record.url?scp=85049487331&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=85049487331&partnerID=8YFLogxK
U2 - 10.1016/j.jmb.2018.06.038
DO - 10.1016/j.jmb.2018.06.038
M3 - Article
C2 - 29944854
AN - SCOPUS:85049487331
VL - 430
SP - 4806
EP - 4820
JO - Journal of Molecular Biology
JF - Journal of Molecular Biology
SN - 0022-2836
IS - 23
ER -