Single molecule fluorescence for membrane proteins

Castell, Oliver K., Dijkman, Patricia M., Wiseman, Daniel and Goddard, Alan (2018). Single molecule fluorescence for membrane proteins. Methods ,

Abstract

The cell membrane is a complex milieu of lipids and proteins. In order to understand the behaviour of individual molecules is it often desirable to examine them as purified components in in vitro systems. Here, we detail the creation and use of droplet interface bilayers (DIBs) which, when coupled to TIRF microscopy, can reveal spatiotemporal and kinetic information for individual membrane proteins. A number of steps are required including modification of the protein sequence to enable the incorporation of appropriate fluorescent labels, expression and purification of the membrane protein and subsequent labelling. Following creation of DIBs, proteins are spontaneously incorporated into the membrane where they can be imaged via conventional single molecule TIRF approaches. Using this strategy, in conjunction with step-wise photobleaching, FRET and / or single particle tracking, a host of parameters can be determined such as oligomerisation state and dynamic information. We discuss advantages and limitations of this system and offer guidance for successful implementation of these approaches.

Publication DOI: https://doi.org/10.1016/j.ymeth.2018.05.024
Divisions: Life & Health Sciences
Life & Health Sciences > Biosciences
Additional Information: © 2018, Elsevier. Licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International http://creativecommons.org/licenses/by-nc-nd/4.0/
Uncontrolled Keywords: Membrane protein,Single molecule fluorescence,Droplet interface bilayer,FRET,Photobleaching,TIRF
Full Text Link:
Related URLs: https://www.sci ... 046202317304334 (Publisher URL)
Published Online Date: 2018-05-30
Authors: Castell, Oliver K.
Dijkman, Patricia M.
Wiseman, Daniel
Goddard, Alan ( 0000-0003-4950-7470)

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Version: Accepted Version

Access Restriction: Restricted to Repository staff only until 30 May 2019.

License: Creative Commons Attribution Non-commercial No Derivatives


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