A User's Guide for Phase Separation Assays with Purified Proteins

Simon Alberti, Shambaditya Saha, Jeffrey B. Woodruff, Titus M. Franzmann, Jie Wang, Anthony A. Hyman

Research output: Contribution to journalArticlepeer-review

111 Scopus citations


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 languageEnglish (US)
Pages (from-to)4806-4820
Number of pages15
JournalJournal of Molecular Biology
Issue number23
StatePublished - Nov 2 2018
Externally publishedYes


  • Sup35
  • low-complexity proteins
  • membrane-less compartment
  • membrane-less organelle
  • phase separation
  • prion-like protein
  • protein purification

ASJC Scopus subject areas

  • Structural Biology
  • Molecular Biology


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