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Single-Molecule Fluorescence Studies of Membrane Transporters Using Total Internal Reflection Microscopy

Journal Article


Abstract


  • Cells are delineated by a lipid bilayer that physically separates the inside from the outer environment. Most polar, charged, or large molecules require proteins to reduce the energetic barrier for passage across the membrane and to achieve transport rates that are relevant for life. Here, we describe techniques to visualize the functioning of membrane transport proteins with fluorescent probes at the single-molecule level. First, we explain how to produce membrane-reconstituted transporters with fluorescent labels. Next, we detail the construction of a microfluidic flow cell to image immobilized proteoliposomes on a total internal reflection fluorescence microscope. We conclude by describing the methods that are needed to analyze fluorescence movies and obtain useful single-molecule data.

Authors


  •   Goudsmits, Joris MH. (external author)
  •   van Oijen, Antoine M.
  •   Slotboom, Dirk J. (external author)

Publication Date


  • 2017

Citation


  • Goudsmits, J. M. H., van Oijen, A. M. & Slotboom, D. J. (2017). Single-Molecule Fluorescence Studies of Membrane Transporters Using Total Internal Reflection Microscopy. Methods in Enzymology, 594 101-121.

Scopus Eid


  • 2-s2.0-85024496957

Number Of Pages


  • 20

Start Page


  • 101

End Page


  • 121

Volume


  • 594

Place Of Publication


  • United States

Abstract


  • Cells are delineated by a lipid bilayer that physically separates the inside from the outer environment. Most polar, charged, or large molecules require proteins to reduce the energetic barrier for passage across the membrane and to achieve transport rates that are relevant for life. Here, we describe techniques to visualize the functioning of membrane transport proteins with fluorescent probes at the single-molecule level. First, we explain how to produce membrane-reconstituted transporters with fluorescent labels. Next, we detail the construction of a microfluidic flow cell to image immobilized proteoliposomes on a total internal reflection fluorescence microscope. We conclude by describing the methods that are needed to analyze fluorescence movies and obtain useful single-molecule data.

Authors


  •   Goudsmits, Joris MH. (external author)
  •   van Oijen, Antoine M.
  •   Slotboom, Dirk J. (external author)

Publication Date


  • 2017

Citation


  • Goudsmits, J. M. H., van Oijen, A. M. & Slotboom, D. J. (2017). Single-Molecule Fluorescence Studies of Membrane Transporters Using Total Internal Reflection Microscopy. Methods in Enzymology, 594 101-121.

Scopus Eid


  • 2-s2.0-85024496957

Number Of Pages


  • 20

Start Page


  • 101

End Page


  • 121

Volume


  • 594

Place Of Publication


  • United States