Determination of sec18-lipid interactions by liposome-binding assay

Matthew L. Starr, Rutilio Fratti

Research output: Chapter in Book/Report/Conference proceedingChapter

Abstract

Protein-lipid binding interactions play a key role in the regulation of peripheral membrane protein function. Liposome-binding assays are a simple and affordable means of screening for specific protein-lipid interactions. Liposomes are prepared by mixing phospholipid combinations of interest before drying and rehydration. Sonication of the lipid mixture produces small unilamellar vesicles (SUVs) which are incubated with a protein of interest to allow for any binding to occur. Liposomes and liposome-protein complexes are floated on a sucrose gradient by centrifugation to separate them from unbound protein. Bound protein levels are easily determined by SDS-PAGE and Western blotting. This approach provides a reliable means of assaying novel protein-lipid interactions in vitro. Here we use liposome floatation to show the binding of the SNARE-activating protein Sec18 (mammalian NSF) to phosphatidic acid.

Original languageEnglish (US)
Title of host publicationMethods in Molecular Biology
PublisherHumana Press Inc.
Pages211-220
Number of pages10
DOIs
StatePublished - Jan 1 2019

Publication series

NameMethods in Molecular Biology
Volume1860
ISSN (Print)1064-3745

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Keywords

  • Liposome
  • Membrane fusion
  • Membrane trafficking
  • NSF
  • Phosphatidic acid
  • Phospholipids
  • SNARE
  • Sec18

ASJC Scopus subject areas

  • Molecular Biology
  • Genetics

Cite this

Starr, M. L., & Fratti, R. (2019). Determination of sec18-lipid interactions by liposome-binding assay. In Methods in Molecular Biology (pp. 211-220). (Methods in Molecular Biology; Vol. 1860). Humana Press Inc.. https://doi.org/10.1007/978-1-4939-8760-3_13