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Single molecule fluorescence for membrane proteins

Castell, Oliver K. ORCID:, Dijkman, Patricia M., Wiseman, Daniel N. and Goddard, Alan D. 2018. Single molecule fluorescence for membrane proteins. Methods 147 , pp. 221-228. 10.1016/j.ymeth.2018.05.024

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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.

Item Type: Article
Date Type: Publication
Status: Published
Schools: Pharmacy
Publisher: Elsevier
ISSN: 1046-2023
Date of First Compliant Deposit: 19 June 2018
Date of Acceptance: 23 May 2018
Last Modified: 24 Oct 2022 16:23

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