Chapter title |
Lipid Exchange by Ultracentrifugation
|
---|---|
Chapter number | 35 |
Book title |
P-Type ATPases
|
Published in |
Methods in molecular biology, January 2016
|
DOI | 10.1007/978-1-4939-3179-8_35 |
Pubmed ID | |
Book ISBNs |
978-1-4939-3178-1, 978-1-4939-3179-8
|
Authors |
Nikolaj Düring Drachmann, Claus Olesen |
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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