Abstract
Lipids play an important role in maintaining P-type ATPase structure and function, and often they are crucial for ATPase activity. When the P-type ATPases are in the membrane, they are surrounded by a mix of different lipid species with varying aliphatic chain lengths and saturation, and the complex interplay between the lipids and the P-type ATPases are still not well understood. We here describe a robust method to exchange the majority of the lipids surrounding the ATPase after solubilisation and/or purification with a target lipid of interest. The method is based on an ultracentrifugation step, where the protein sample is spun through a dense buffer containing large excess of the target lipid, which results in an approximately 80–85 % lipid exchange. The method is a very gently technique that maintains protein folding during the process, hence allowing further characterization of the protein in the presence of a target lipid of interest.
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Acknowledgement
We would like to thank Jesper Vuust Møller for comments and discussion in the preparation of this book chapter.
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Drachmann, N.D., Olesen, C. (2016). Lipid Exchange by Ultracentrifugation. In: Bublitz, M. (eds) P-Type ATPases. Methods in Molecular Biology, vol 1377. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-3179-8_35
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DOI: https://doi.org/10.1007/978-1-4939-3179-8_35
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-3178-1
Online ISBN: 978-1-4939-3179-8
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