Isolation and Analysis of Detergent-Resistant Membrane Fractions

Abstract

The hypothesis that the Golgi apparatus is capable of sorting proteins and sending them to the plasma membrane through "lipid rafts," membrane lipid domains highly enriched in glycosphingolipids, sphingomyelin, ceramide, and cholesterol, was formulated by van Meer and Simons in 1988 and came to a turning point when it was suggested that lipid rafts could be isolated thanks to their resistance to solubilization by some detergents, namely Triton X-100. An incredible number of papers have described the composition and properties of detergent-resistant membrane fractions. However, the use of this method has also raised the fiercest criticisms. In this chapter, we would like to discuss the most relevant methodological aspects related to the preparation of detergent-resistant membrane fractions, and to discuss the importance of discriminating between what is present on a cell membrane and what we can prepare from cell membranes in a laboratory tube.

Cathepsin Inhibition Prevents Autophagic Protein Turnover and Downregulates Insulin Growth Factor-1 Receptor-Mediated Signaling in Neuroblastoma

“…Our standard cell protein solubilization technique using urea and thiourea prevents loss of these triton-insoluble proteins. Proteins in the cytoskeleton, in lipid rafts and in protein aggregates, have all been reported to be poorly extracted by Triton-X-100 (Vale et al, 1985;Fujita et al, 2007;Aureli et al, 2016). We are not aware of prior reports on accumulation of Triton-insoluble fragments of the IGF-1 receptor after protease inhibition.…”

Sphingolipids and plasma membrane hydrolases in human primary bronchial cells during differentiation and their altered patterns in cystic fibrosis

“…We addressed this issue exploiting the strategy that allows to obtain an enrichment of apical membrane portions based on biotin-streptavidin precipitation of all the proteins residing in the outer leaflet of the apical membrane in lipid-domain preserving conditions [ 26 ]. HBE-WT and HBE-ΔF cells fully differentiated at ALI were fed from the apical and basolateral side with [1- 3 H]sphingosine to metabolically label at the steady state cell SLs.…”

Linker length in fluorophore–cholesterol conjugates directs phase selectivity and cellular localisation in GUVs and live cells

“…Although widely distributed in the cell membrane, cholesterol is well-established as an essential component of detergent resistant membrane domains in cells and by extension in lipid ras. 52 In model membranes, cholesterol has been shown to promote formation of L o domains and partitions with greater preference into these domains than the L d . 53,54 The origin of cholesterol preference for L o and its condensing effect on the membrane has been discussed widely in the literature.…”