Encyclopedia of Experiments: Biological Techniques
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Transcript
Protein-phospholipid interactions on the cell membrane are crucial for the regulation of several cellular processes.
To detect target protein-phospholipid interactions in vitro using quantitative flow cytometry, take a tube containing a desired concentration of cell membrane mimic — micron-sized, lipophilic dye-labeled phospholipid vesicles — in buffer.
Add an appropriate concentration of different fluorescently-labeled target proteins in solution.
Inject the suspension into the flow cytometer. Set the fluidics system parameters to a low flow rate to allow sufficient time for the protein-phospholipid interactions.
As the fluorescently-labeled proteins and phospholipid vesicles interact in the flow channel, the concentration of proteins bound to the vesicles increases.
As these complexes pass through the laser beams from specific fluorescence channels providing appropriate wavelengths, they become excited. These excited proteins and phospholipid vesicles in the complex emit respective fluorescence signals upon relaxation to the ground state, which are captured by the appropriate detectors.
The detection of a high mean fluorescence intensity of the proteins is indicative of protein binding to the phospholipid vesicles.
