title: Lipid Exchange by Ultracentrifugation.
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.
authors.affiliation: Centre for Membrane Pumps in Cells and Disease - PUMPkin, Danish National Research Foundation, Aarhus, Denmark. ndd06@mb.au.dk.|Syngenta, Jealott's Hill International Research Centre, Bracknell, Berkshire, RG42 6EY, United Kingdom. ndd06@mb.au.dk.
authors.forename: Nikolaj Düring
authors.initials: ND
authors.lastname: Drachmann
authors.affiliation: Centre for Membrane Pumps in Cells and Disease - PUMPkin, Danish National Research Foundation, Aarhus, Denmark.|Department of Biomedicine, Aarhus University, Aarhus, Denmark.
authors.forename: Claus
authors.initials: C
authors.lastname: Olesen
mesh_terms: D053000:Analytic Sample Preparation Methods; D000818:Animals; D010713:Phosphatidylcholines / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D011817:Rabbits; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases / Q000737:chemistry / Q000378:metabolism; D012995:Solubility; D014461:Ultracentrifugation / Q000379:methods*
chemical_list: D010713:Phosphatidylcholines; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; C017251:1,2-oleoylphosphatidylcholine
keywords: Density gradient; Lipid exchange; Native lipids; Synthetic lipids; Ultracentrifugation