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Single Liposome Measurements for the Study of Proton-Pumping Membrane Enzymes Using Electrochemistry and Fluorescent Microscopy
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Biochemistry
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JoVE Journal Biochemistry
Single Liposome Measurements for the Study of Proton-Pumping Membrane Enzymes Using Electrochemistry and Fluorescent Microscopy

Single Liposome Measurements for the Study of Proton-Pumping Membrane Enzymes Using Electrochemistry and Fluorescent Microscopy

DOI:
10.3791/58896-v

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12:15 min

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February 21, 2019

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, ,
1School of Biomedical Sciences & the Astbury Centre for Structural Molecular Biology,University of Leeds, 2Department of Chemistry and Nano-Science Center,University of Copenhagen

Chapters

  • 00:04Title
  • 00:28Preparation of a Lipid/UQ-10/FDLL Mix
  • 01:26Reconstitution of Cytochrome bo3
  • 05:06Fabrication of Semi-Transparent Gold Electrodes and Modification of the Gold Surface with Self-Assembled Monolayer (SAM)
  • 06:07Electrochemical Testing of Proteoliposomes Activity
  • 07:46Detection of Enzymatic Proton Pumping by Fluorescence Microscopy
  • 09:44Results: Liposome Identification and pH Change
  • 11:35Conclusion

Summary

Automatic Translation

Automatic Translation

Here, we present a protocol to study the molecular mechanism of proton translocation across lipid membranes of single liposomes, using cytochrome bo3 as an example. Combining electrochemistry and fluorescence microscopy, pH changes in the lumen of single vesicles, containing single or multiple enzyme, can be detected and analyzed individually.

Tags

Keywords: Single LiposomeProton-pumpingMembrane EnzymesElectrochemistryFluorescent MicroscopyCytochrome BO3Escherichia ColiUbiquinone 10FDLLMOPS BufferHPTSOGP SurfactantPolystyrene Microbeads

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