Single molecule fluorescence for membrane proteins

Oliver K. Castell, Patricia M. Dijkman, Daniel Wiseman, Alan Goddard

Research output: Contribution to journalArticlepeer-review


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.
Original languageEnglish
Pages (from-to)221-228
Number of pages8
Early online date30 May 2018
Publication statusPublished - 1 Sept 2018

Bibliographical note

© 2018, Elsevier. Licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International


  • Membrane protein
  • Single molecule fluorescence
  • Droplet interface bilayer
  • FRET
  • Photobleaching
  • TIRF


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