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Journal of Lipid Research, Vol. 50, 1245-1254, June 2009
Copyright © 2009 by American Society for Biochemistry and Molecular Biology

Methods

A novel flow cytometric assay to quantify interactions between proteins and membrane lipids

Koen Temmerman and Walter Nickel¹

Heidelberg University Biochemistry Center, 69120 Heidelberg, Germany

This study was supported by the Collaborative Research Center 638 of the German Research Foundation.

Published, JLR Papers in Press, January 14, 2009.

¹ To whom correspondence should be addressed. e-mail: walter.nickel{at}bzh.uni-heidelberg.de

A diverse set of experimental systems has been developed to probe protein-lipid interactions. These include measurements with the headgroups of membrane lipids in solution, immobilized membrane lipids, and analysis of protein binding to membrane lipids reconstituted in liposomes. Each of these methodologies has strengths but also substantial limitations. For example, measurements between proteins and lipid headgroups or with immobilized membrane lipids do not probe interactions in their natural environment, the lipid bilayer. The use of liposomes, however, was so far mostly restricted to biochemical flotation experiments that do not provide quantitative and/or kinetic data. Here, we present a fast and sensitive flow cytometric method to detect protein-lipid interactions. This technique allows for quantitative measurements of interactions between multiple fluorescently labeled proteins and membrane lipids reconstituted in lipid bilayers. The assay can be used to quantify binding efficiencies and to determine kinetic constants. The method is further characterized by a short sampling time of only a few seconds that allows for high-content screening procedures. Finally, using light scatter measurements, the described method also allows for monitoring changes of membrane curvature as well as tethering of liposomes evoked by binding of proteins.

Supplementary key words method • quantitative • fluorescence-activated cell sorter • liposome • phosphoinositide • lipid binding • Pleckstrin Homology domain • Fibroblast Growth Factor 2 • competitive binding • high-throughput

Abbreviations: (c.)F.U., (corrected) fluorescence units; FACS, fluorescence-activated cell sorter; FGF-2, Fibroblast Growth Factor 2; GFP, green fluorescent protein; ITC, isothermal titration calorimetry; K_d, dissociation constant; PH-PLC₁, Pleckstrin Homology domain of Phospolipase C-₁; PI(4,5)P₂, phosphatidylinositol-4,5-bisphosphate; rhod-PE, rhodamine-labeled phosphatidylethanolamine; SPR, surface plasmon resonance

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