A Spectrophotometric Turbidity Assay to Study Liquid-Liquid Phase Separation of UBQLN2 In Vitro

Peter Raymond-Smiedy, Barrington Bucknor, Yiran Yang, Tongyin Zheng, Carlos A. Castañeda

Research output: Chapter in Book/Report/Conference proceedingChapter

Abstract

Liquid–liquid phase separation (LLPS) is hypothesized to be the underlying mechanism for how membraneless organelles or biomolecular condensates form inside both prokaryotic and eukaryotic cells. Protein LLPS is a biophysical process during which proteins demix from homogeneous solution to form protein-dense droplets with liquid-like properties. Disruptions to LLPS, such as changes to material properties of condensates or physicochemical parameters for LLPS onset, are implicated in neurodegenerative diseases and cancer. Therefore, it is essential to determine the physicochemical parameters that promote protein LLPS. Here, we present our UV-Vis spectrophotometric turbidity assay to characterize the temperature and concentration dependence of LLPS for UBQLN2, a protein that undergoes LLPS via homotypic interactions in vitro and forms stress-induced condensates in cells. Mutations in UBQLN2 cause amyotrophic lateral sclerosis (ALS) and disrupt UBQLN2 LLPS. We present a detailed expression and purification protocol for a C-terminal construct of UBQLN2 and how we use microscopy to image UBQLN2 LLPS. We use our UV-Vis assay to construct temperature–concentration phase diagrams for wild-type and mutant UBQLN2 constructs to determine the effects of domain deletions and/or mutations on UBQLN2 phase separation.

Original languageEnglish (US)
Title of host publicationMethods in Molecular Biology
PublisherHumana Press Inc.
Pages515-541
Number of pages27
DOIs
StatePublished - 2023

Publication series

NameMethods in Molecular Biology
Volume2551
ISSN (Print)1064-3745
ISSN (Electronic)1940-6029

Keywords

  • LCST
  • Liquid-liquid phase separation
  • Microscopy
  • Phase diagrams
  • Phase transitions
  • Spectrophotometric assay
  • Turbidity
  • UBQLN2
  • UCST

ASJC Scopus subject areas

  • Molecular Biology
  • Genetics

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