Isolating and Analyzing Protein Containing Granules from Cells

Rachel A. Victor, Valery F. Thompson, Jacob C. Schwartz

Research output: Contribution to journalArticlepeer-review

Abstract

Recent advancements in detection methods have made protein condensates, also called granules, a major area of study, but tools to characterize these assemblies need continued development to keep up with evolving paradigms. We have optimized a protocol to separate condensates from cells using chemical cross-linking followed by size-exclusion chromatography (SEC). After SEC fractionation, the samples can be characterized by a variety of approaches including enzyme-linked immunosorbent assay, dynamic light scattering, electron microscopy, and mass spectrometry. The protocol described here has been optimized for cultured mammalian cells and E. coli expressing recombinant proteins. Since the lysates are fractionated by size, this protocol can be modified to study other large protein assemblies, including the nuclear pore complex, and for other tissues or organisms.

Original languageEnglish (US)
Article numbere35
JournalCurrent Protocols
Volume1
Issue number3
DOIs
StatePublished - Mar 2021

Keywords

  • condensates
  • formaldehyde crosslinking
  • granule
  • liquid-liquid phase separation
  • non-membrane bound organelles
  • protein assemblies
  • size-exclusion chromatography

ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)
  • Pharmacology, Toxicology and Pharmaceutics(all)
  • Neuroscience(all)
  • Immunology and Microbiology(all)
  • Health Informatics
  • Medical Laboratory Technology

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