A convenient protocol for generating giant unilamellar vesicles containing SNARE proteins using electroformation

Reconstitution of membrane proteins in artificial membranes is an essential prerequisite for functional studies that depend on the context of an intact membrane. While straight-forward protocols for reconstituting proteins in small unilamellar vesicles were developed many years ago, it is much more...

Full description

Saved in:
Bibliographic Details
Published in:Scientific reports 2018-06, Vol.8 (1), p.9422-8, Article 9422
Main Authors: Witkowska, Agata, Jablonski, Lukasz, Jahn, Reinhard
Format: Article
Language:English
Subjects:
14/19
14/63
631/1647/2204
631/57/2271
Electronic equipment
Exocytosis
Humanities and Social Sciences
Membrane proteins
Membranes
multidisciplinary
Proteins
Science
Science (multidisciplinary)
SNAP receptors
Synaptic vesicles
Citations: Items that this one cites
Items that cite this one
Online Access:Get full text
Tags: Add Tag
No Tags, Be the first to tag this record!
Description
Summary:Reconstitution of membrane proteins in artificial membranes is an essential prerequisite for functional studies that depend on the context of an intact membrane. While straight-forward protocols for reconstituting proteins in small unilamellar vesicles were developed many years ago, it is much more difficult to prepare large membranes containing membrane proteins at biologically relevant concentrations. Giant unilamellar vesicles (GUVs) represent a model system that is characterised by low curvature, controllable tension, and large surface that can be easily visualised with microscopy, but protein insertion is notoriously difficult. Here we describe a convenient method for efficient generation of GUVs containing functionally active SNARE proteins that govern exocytosis of synaptic vesicles. Preparation of proteo-GUVs requires a simple, in-house-built device, standard and inexpensive electronic equipment, and employs a straight-forward protocol that largely avoids damage of the proteins. The procedure allows upscaling and multiplexing, thus providing a platform for establishing and optimizing preparation of GUVs containing membrane proteins for a diverse array of applications.
ISSN:2045-2322
2045-2322
DOI:10.1038/s41598-018-27456-4