Elucidating Membrane Structure and Protein Behavior Using Giant Plasma Membrane Vesicles

Overview

Journal Nat Protoc

Specialties Biology
Pathology
Science

Date 2012 May 5

PMID 22555243

Citations 243

Authors

Erdinc Sezgin

Hermann-Josef Kaiser

Tobias Baumgart

Petra Schwille

Kai Simons

Ilya Levental

Affiliations

Soon will be listed here.

Abstract

The observation of phase separation in intact plasma membranes isolated from live cells is a breakthrough for research into eukaryotic membrane lateral heterogeneity, specifically in the context of membrane rafts. These observations are made in giant plasma membrane vesicles (GPMVs), which can be isolated by chemical vesiculants from a variety of cell types and microscopically observed using basic reagents and equipment available in any cell biology laboratory. Microscopic phase separation is detectable by fluorescent labeling, followed by cooling of the membranes below their miscibility phase transition temperature. This protocol describes the methods to prepare and isolate the vesicles, equipment to observe them under temperature-controlled conditions and three examples of fluorescence analysis: (i) fluorescence spectroscopy with an environment-sensitive dye (laurdan); (ii) two-photon microscopy of the same dye; and (iii) quantitative confocal microscopy to determine component partitioning between raft and nonraft phases. GPMV preparation and isolation, including fluorescent labeling and observation, can be accomplished within 4 h.

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