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SNAREs pp 211-220 | Cite as

Determination of Sec18-Lipid Interactions by Liposome-Binding Assay

  • Matthew L. Starr
  • Rutilio Fratti
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1860)

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.

Key words

Liposome Phospholipids Membrane trafficking Membrane fusion Sec18 NSF Phosphatidic acid SNARE 

Notes

Acknowledgments

This work was supported in part by NIH grant GM101132 to RAF.

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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Department of BiochemistryUniversity of Illinois at Urbana-ChampaignUrbanaUSA

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