The single-molecule approach to membrane protein stoichiometry

Michael G. Nichols, Richard J. Hallworth

Research output: Chapter in Book/Report/Conference proceedingChapter

1 Citation (Scopus)

Abstract

The advent of techniques for imaging solitary fluorescent molecules has made possible many new kinds of biological experiments. Here, we describe the application of single-molecule imaging to the problem of subunit stoichiometry in membrane proteins. A membrane protein of unknown stoichiometry, prestin, is coupled to the fluorescent enhanced green fluorescent protein (eGFP) and synthesized in the human embryonic kidney (HEK) cell line. We prepare adherent membrane fragments containing prestin-eGFP by osmotic lysis. The molecules are then exposed to continuous low-level excitation until their fluorescence reaches background levels. Their fluorescence decreases in discrete equal-amplitude steps, consistent with the photobleaching of single fluorophores. We count the number of steps required to photobleach each molecule. The molecular stoichiometry is then deduced using a binomial model.

Original languageEnglish (US)
Title of host publicationMethods in Molecular Biology
PublisherHumana Press Inc.
Pages189-199
Number of pages11
Volume1427
DOIs
StatePublished - 2016

Publication series

NameMethods in Molecular Biology
Volume1427
ISSN (Print)10643745

Fingerprint

Membrane Proteins
Fluorescence
Photobleaching
Statistical Models
Kidney
Cell Line
Membranes
enhanced green fluorescent protein
Single Molecule Imaging

All Science Journal Classification (ASJC) codes

  • Molecular Biology
  • Genetics

Cite this

Nichols, M. G., & Hallworth, R. J. (2016). The single-molecule approach to membrane protein stoichiometry. In Methods in Molecular Biology (Vol. 1427, pp. 189-199). (Methods in Molecular Biology; Vol. 1427). Humana Press Inc.. https://doi.org/10.1007/978-1-4939-3615-1_11

The single-molecule approach to membrane protein stoichiometry. / Nichols, Michael G.; Hallworth, Richard J.

Methods in Molecular Biology. Vol. 1427 Humana Press Inc., 2016. p. 189-199 (Methods in Molecular Biology; Vol. 1427).

Research output: Chapter in Book/Report/Conference proceedingChapter

Nichols, MG & Hallworth, RJ 2016, The single-molecule approach to membrane protein stoichiometry. in Methods in Molecular Biology. vol. 1427, Methods in Molecular Biology, vol. 1427, Humana Press Inc., pp. 189-199. https://doi.org/10.1007/978-1-4939-3615-1_11
Nichols MG, Hallworth RJ. The single-molecule approach to membrane protein stoichiometry. In Methods in Molecular Biology. Vol. 1427. Humana Press Inc. 2016. p. 189-199. (Methods in Molecular Biology). https://doi.org/10.1007/978-1-4939-3615-1_11
Nichols, Michael G. ; Hallworth, Richard J. / The single-molecule approach to membrane protein stoichiometry. Methods in Molecular Biology. Vol. 1427 Humana Press Inc., 2016. pp. 189-199 (Methods in Molecular Biology).
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