Naslov
Methods for determining ganglioside distributions in lipid rafts

Autori
Mlinac Jerković, Kristina ; Ilić, Katarina ; Damjanović, Vladimir ; Kalanj Bognar, Svjetlana ; Schnaar, Ronald L. ; Heffer, Marija

Vrsta, podvrsta i kategorija rada
Sažeci sa skupova, sažetak, znanstveni

Izvornik
Glycobiology / - : Oxford Journals, 2016, 112-113

Skup
Society for glycobiology 2016 Annual Meeting

Mjesto i datum
New Orleans (LA), Sjedinjene Američke Države, 19.11.2016. - 22.11.2016

Vrsta sudjelovanja
Poster

Vrsta recenzije
Međunarodna recenzija

Ključne riječi
Lipid rafts; gangliosides; Brij O20

Sažetak
Lipid rafts are dynamic membrane microdomains with distinct lipid and protein compositions. They are involved in regulation of membrane functions and serve as signaling platforms of the cell. There is an increasing interest in the investigation of lipid raft dynamics and their precise compositions due to the established role of lipid rafts in pathogenesis of numerous human disorders. Various approaches to isolating lipid rafts have been used to separate them from the bulk (non-raft) membranes. Most often, detergent- resistant membranes (DRMs) enriched in cholesterol and sphingomyelin and thought to be related to rafts, are extracted using the non- ionic detergent Triton X-100 (Tx-100). However, Tx-100 causes a major redistribution of membrane gangliosides and is therefore unacceptable as a detergent for lipid raft isolation for studying gangliosides and ganglioside-associated proteins. Published non- detergent methods for lipid raft isolation were not sufficiently effective or reproducible in our hands. In this work lipid rafts were isolated from bulk lipids by density centrifugation after membrane solubilization using a detergent that does not result in redistribution of gangliosides in membranes: Brij O20 (previously known as Brij 98). Successful raft isolation was confirmed by Western blot analysis of the lipid raft marker protein flotilin and the non-lipid raft membrane protein transferrin receptor and the method was found to be highly reproducible. Since the aim of this work is to develop a method for ganglioside purification from isolated lipid rafts for subsequent analytical studies, several methods were then tested to solubilize and separate gangliosides from excess of Brij O20. Brij O20 is polyoxyethylene ether with a molecular weight of 1150 Da (similar size to gangliosides), and readilly forms micelles (including mixed micelles with gangliosides). We employed several purification techniques, including organic solvent extraction, ion-exchange chromatography and adsorption-partition chromatography. The outcome was assessed by HPTLC. Organic solvent extraction and ion- exchange chromatography were not efficient enough in separating Brij O20 from gangliosides. Better separation was achieved by adsorption-partition chromatography, however further optimization is needed. This work will enable more detailed lipid raft analysis in respect to ganglioside composition and structure and lead to improved resolution of lipid-protein relations within lipid rafts.

Izvorni jezik
Engleski

Znanstvena područja
Temeljne medicinske znanosti

POVEZANOST RADA