Microscale Thermophoresis to Study Protein-Lipid Interactions in Solution

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.

Before beginning an analysis, turn on the power switch on the back of the MST device, and open the control software. Confirm that the computer is attached to the device and in "Connected" status, and set the MST Before to 3 seconds, the MST to 30 seconds, and the Fluorescence Recovery to after 1 second.

For each capillary tube, enter the name of the target ligand, the name of the ligand analyte, the concentration of the target, and the highest titration concentration using the autofill titration ratio. Select a range of MST powers and enter the values for each power to test for the most robust binding fit.

To prepare labeled MST samples, dilute a Vam7-octahistidine solution to a 200-nanomolar concentration and an NTA-Atto 647 dye to a 100-nanomolar concentration, both in PBS. Mix the Vam7-octahistidine and the NTA-Atto 647 dye at a 1 to 1 volumetric ratio, and allow the mixture to sit at room temperature, protected from light, for 30 minutes. At the end of the incubation, centrifuge the dye and protein mixture, and store the solution at 4 degrees Celsius for up to a few hours before use. Then, prepare the sample. Take dye to ligand and expose the analyte.

For MicroScale Thermophoresis of the sample, open the device and slide the capillary rack out. Load the sample capillaries into the rack with the highest concentration at position 1, and select the red channel in the control software. Then, load the rack in the instrument. Then, select a range of MST power and start the Cap Scan + MST measurement and evaluate the shift response.