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Lipid-Protein Interactions pp 233–252Cite as

A Guide to Tracking Single Transmembrane Proteins in Supported Lipid Bilayers

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Part of the book series: Methods in Molecular Biology ((MIMB,volume 974))

Abstract

The purpose of this chapter is to serve as a guide for those who wish to carry out experiments tracking single transmembrane proteins in planar supported membrane biomimetics. This chapter describes, in detail, the construction of a simple single-molecule microscope, which includes (1) a parts list, (2) an alignment procedure, (3) a calibration procedure, and (4) a procedure for measuring the mechanical stability of the instrument. It also gives procedures for making planar supported POPC bilayers on hydrophilically treated borosilicate and quartz, POPC/PEG-PE cushioned bilayers on hydrophilically treated surfaces, and POPC/PEG-PE cushioned bilayers on BSA passivated substrates. The procedure for the detergent-mediated incorporation of the transmembrane protein 5HT3A (a serotonin receptor) is also described and can be used as a starting point for other large non-self-inserting transmembrane proteins. The final experimental section of this chapter details different procedures for data analysis including (1) a quantitative analysis of mean displacements from individually tracked particles, (2) a Gaussian analysis of step-size distributions, (3) the Gaussian analysis of diffusion coefficients from ensembles of transmembrane proteins, and (4) a perspective associated with the interpretation of single-particle tracking data.

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Authors and Affiliations

  1. Department of Chemistry, Washington State University, Pullman, WA, USA

    Kumud Raj Poudel, Jeffrey P. Jones & James A. Brozik

Authors
  1. Kumud Raj Poudel
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  2. Jeffrey P. Jones
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  3. James A. Brozik
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Corresponding author

Correspondence to James A. Brozik .

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Editors and Affiliations

  1. Institut für Biologie, Abteilung Biophysik, Universität Kassel, Heinrich-Plett-Straße 40, Kassel, 34132, Germany

    Jörg H. Kleinschmidt

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Poudel, K.R., Jones, J.P., Brozik, J.A. (2013). A Guide to Tracking Single Transmembrane Proteins in Supported Lipid Bilayers. In: Kleinschmidt, J. (eds) Lipid-Protein Interactions. Methods in Molecular Biology, vol 974. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-275-9_11

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  • DOI: https://doi.org/10.1007/978-1-62703-275-9_11

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  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-62703-274-2

  • Online ISBN: 978-1-62703-275-9

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