Lipid Extraction from HeLa Cells, Quantification of Lipids, Formation of Large Unilamellar Vesicles (LUVs) by Extrusion and in vitro Protein-lipid Binding Assays, Analysis of the Incubation Product by Transmission Electron Microscopy (TEM) and by Flotation across a Discontinuous Sucrose Gradient
Amina Bittame,
Jodie Lopez,
Gregory Effantin,
Nicolas Blanchard,
Marie-France Cesbron-Delauw,
Jean Gagnon,
Corinne Mercier
Affiliations
Amina Bittame
Labroatoire Adaptation et Pathogénie des Microorganismes (LAPM), Grenoble, FranceCNRS UMR 5163, Grenoble, France, Université Grenoble Alpes, Grenoble, France
Jodie Lopez
Centre de Physiopathologie de Toulouse-Purpan (CPTP), Toulouse, FranceINSERM UMR 1043, Toulouse, France, CNRS UMR 5282, Toulouse, France, Université de Toulouse III, Toulouse, France
Gregory Effantin
Université Grenoble Alpes, Grenoble, FranceInstitut de Biologie Structurale (IBS), Grenoble, France, CNRS UMR 5075, Grenoble, France, Commissariat à l’Energie Atomique (CEA), Grenoble, France
Nicolas Blanchard
Centre de Physiopathologie de Toulouse-Purpan (CPTP), Toulouse, FranceINSERM UMR 1043, Toulouse, France, CNRS UMR 5282, Toulouse, France, Université de Toulouse III, Toulouse, France
Marie-France Cesbron-Delauw
Université Grenoble Alpes, Grenoble, FranceLabroatoire Techniques de l’Ingénierie Médicale et de la Complexité - Informatique, Mathématiques et Applications (TIMC-IMAG), Grenoble, France, CNRS UMR 5525, Grenoble, France
Jean Gagnon
Labroatoire Adaptation et Pathogénie des Microorganismes (LAPM), Grenoble, FranceCNRS UMR 5163, Grenoble, France, Université Grenoble Alpes, Grenoble, France
Corinne Mercier
Université Grenoble Alpes, Grenoble, FranceLabroatoire Techniques de l’Ingénierie Médicale et de la Complexité - Informatique, Mathématiques et Applications (TIMC-IMAG), Grenoble, France, CNRS UMR 5525, Grenoble, France
Dissecting the interactions established between proteins and membranes in a given type of cells is not an easy task. Using a cell-free system of large unilamellar vesicles (LUVs) to analyze these interactions may help decipher these interactions and identify potential membrane deformations induced by the proteins incubated with these LUVs. This article describes the protocols for 1) extraction of total lipids from eukaryotic cells using the method developed by Bligh and Dyer (1959), 2) the quantification of glycerophospholipids by gas chromatography after methanolysis, followed by 3) the formation of LUVs by extrusion, 4) protein-lipid binding assay, 5) analysis of the incubation product by transmission electron microscopy (TEM) and by flotation across a discontinuous sucrose gradient and finally, 6) analysis of the proteins by immunoblot and revelation of the glycerophospholipids by iodin fumigation.
