JoVE Journal

Biochemistry

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

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.

Capitoli in questo video

0:04

Title

0:28

Preparation of a Lipid/UQ-10/FDLL Mix

1:26

Reconstitution of Cytochrome bo3

5:06

Fabrication of Semi-Transparent Gold Electrodes and Modification of the Gold Surface with Self-Assembled Monolayer (SAM)

6:07

Electrochemical Testing of Proteoliposomes Activity

7:46

Detection of Enzymatic Proton Pumping by Fluorescence Microscopy

9:44

Results: Liposome Identification and pH Change

11:35

Conclusion

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