TY  - JOUR
TI  - 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
AU  - Bittame, Amina 
AU  - Lopez, Jodie 
AU  - Effantin, Gregory 
AU  - Blanchard, Nicolas 
AU  - Cesbron-Delauw, Marie-France 
AU  - Gagnon, Jean  
AU  - Mercier, Corinne 
VL  - 6
IS  - 20
PY  - 2016
DA  - 2016/10/20
SP  - e1963
C1  - Bio-protocol 2016;6:e1963
DO  - 10.21769/BioProtoc.1963
UR  - https://doi.org/10.21769/BioProtoc.1963
AB  - 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.
KW  - Large Unilamellar Vesicles (LUVs)
KW  - Liposomes
KW  - Protein-Lipid Binding Assay
KW  - Discontinuous Sucrose Gradients
KW  - Transmission Electron Microscopy
JF  - Bio-protocol
SN  - 2331-8325
PB  - Bio-protocol LLC.
BIO101  - False