The activity of many biomolecules and drugs crucially depends on whether they bind to biological membranes and whether they translocate to the opposite lipid leaflet and trans aqueous compartment. A general strategy to measure membrane binding and permeation is the uptake and release assay, which compares two apparent equilibrium situations established either by the addition or by the extraction of the solute of interest. Only solutes that permeate the membrane sufficiently fast do not show any dependence on the history of sample preparation. This strategy can be pursued for virtually all membrane-binding solutes, using any method suitable for detecting binding. Here, we present in detail one example that is particularly well developed, namely the nonspecific membrane partitioning and flip-flop of small, nonionic solutes as characterized by isothermal titration calorimetry. A complete set of experiments, including all sample preparation procedures, can typically be accomplished within 2 days. Analogous protocols for studying charged solutes, virtually water-insoluble, hydrophobic compounds or specific ligands are also considered.
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We dedicate this publication to Professor Joachim Seelig of the Biozentrum of the University of Basel, on the occasion of his 65th birthday. He has played a seminal role in the development of membrane microcalorimetry. We are indebted to him for the friendly uptake into his laboratory, his invaluable advice and support and the generous release into the freedom to pursue our own projects and plans. We thank Natalie Bordag for help with compiling the fitting routine. This work was supported by a fellowship within the Postdoc Program of the German Academic Exchange Service (DAAD) to A.D.T., by grant no. QLK3-CT-2002-01989 of the European Commission to S.K. and by grant no. 31-67216.01 of the Swiss National Science foundation to H.H.
The authors declare no competing financial interests.
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Tsamaloukas, A., Keller, S. & Heerklotz, H. Uptake and release protocol for assessing membrane binding and permeation by way of isothermal titration calorimetry. Nat Protoc 2, 695–704 (2007) doi:10.1038/nprot.2007.98
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