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SNAREs pp 211–220Cite as

Determination of Sec18-Lipid Interactions by Liposome-Binding Assay

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

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Acknowledgments

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

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Correspondence to Rutilio Fratti .

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Starr, M.L., Fratti, R. (2019). Determination of Sec18-Lipid Interactions by Liposome-Binding Assay. In: Fratti, R. (eds) SNAREs. Methods in Molecular Biology, vol 1860. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-8760-3_13

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  • DOI: https://doi.org/10.1007/978-1-4939-8760-3_13

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-8759-7

  • Online ISBN: 978-1-4939-8760-3

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