Encyclopedia of Experiments: Biological Techniques
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Transcript
To study the interaction of proteins with lipid molecules, begin with two capillary tubes. Add fluorescent dye-labeled test proteins in both tubes and unlabeled lipid molecules to one of them. Place these capillaries in an array and place them inside a microscale thermophoresis instrument.
Irradiate the protein-containing capillary with focused infrared light, creating a temperature rise in the focal spot that diffuses through the solution, generating a temperature gradient within a narrow region containing fluorescently-labeled proteins.
The fluorescence detector collects the fluorescence signal from the same spot of the capillary — where the laser is focused. The processor converts the signal and generates a plot of the relative fluorescence as a function of time.
In a temperature gradient, labeled proteins start migrating from the hotter region to the cooler region, exhibiting thermophoresis — a phenomenon of temperature-dependent migration.
Due to thermophoresis, the fluorescence intensity gradually decreases over time and attains a steady state, where labeled proteins reach equilibration.
Irradiate the protein-lipid mix-containing capillary, creating a temperature gradient.
During thermophoresis, the migrating labeled proteins interact with the lipid molecules, forming lipid-protein complexes with higher molecular masses. This leads to retardation of their movement.
This differential migration causes a shift in fluorescence, confirming successful protein-lipid interaction.
