Abstract
Protein–lipid interactions play an important role in cellular protein relocation, activation and signal transduction. The liposome-binding assay is a simple and inexpensive method to examine protein–lipid binding in vitro. The phospholipids used for liposome production are dried and hydrated. Subsequent extrusion of the phospholipid mixture ensures the production of large unilamellar vesicles (LUV) filled with raffinose. Those LUVs can be easily separated from the aqueous solution by centrifugation. By incubating a protein of interest with the LUVs and subsequent centrifugation steps, the bound protein fraction can be determined using Western Blot or Coomassie staining. This technique enables analysis of protein–lipid binding affinity and specificity.
Key words
- Large unilamellar vesicle (LUV)
- Liposome assay
- Phospholipid binding
- Binding affinity
- Phosphatidic acid
- Phospholipid signaling
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Acknowledgments
The authors thank Carlos Galvan-Ampudia for his help in cloning the PH-domain of AtPDK1 in the GST expression vector and Edgar Kooijman for support and advice on lipid physiochemical properties. This work was supported by the Netherlands Organisation for Scientific Research (NWO) grants Vidi 700.56.429 and ALW 820.02.017, STW Perspectief 10987, NGI Horizon project 93511011, NSF Grants DMR-0844115 and CHE-0922848, Kent State, and an ICAM fellowship to JMR (ICAM Branches Cost Sharing Fund). Financial support from EU-COST Action FA0605 is also gratefully acknowledged.
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Julkowska, M.M., Rankenberg, J.M., Testerink, C. (2013). Liposome-Binding Assays to Assess Specificity and Affinity of Phospholipid–Protein Interactions. In: Munnik, T., Heilmann, I. (eds) Plant Lipid Signaling Protocols. Methods in Molecular Biology, vol 1009. Humana, Totowa, NJ. https://doi.org/10.1007/978-1-62703-401-2_24
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DOI: https://doi.org/10.1007/978-1-62703-401-2_24
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