Determination of Sec18-Lipid Interactions by Liposome-Binding Assay.

Matthew L Starr, Rutilio Fratti

Methods in molecular biology (Clifton, N.J.) 2019

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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.

Citation

Matthew L Starr, Rutilio Fratti. Determination of Sec18-Lipid Interactions by Liposome-Binding Assay. Methods in molecular biology (Clifton, N.J.). 2019;1860:211-220