Single-Molecule Imaging to Visualize Fusion Protein Condensates Assembly on DNA Curtains
成績單
To image the EWS-FLI1 condensation formation on DNA curtains, open the imaging software and find and mark the positions of the nine zig-zag patterns under bright-field. Then, turn on the flow at 0.2 milliliters per minute to stain the DNA with double-stranded DNA dye for 10 minutes.
Next, dilute the mCherry-EWS-FLI1 protein with the imaging buffer at a concentration of 100 nanomoles and 100 microliters. Then, load the protein sample through the valve with a 100-microliter glass syringe, and change the flow rate to 0.4 milliliters per minute.
After turning on the 488-nanometer laser, pre-scan each region to check the DNA distribution state, and select the region in which the DNA molecules distribute evenly. Then, set the laser power to 10% for the 488-nanometer laser and 20% for the 561-nanometer laser, and using the power meter, measure the real laser power near the prism.
Start acquiring images at two-second intervals with both 488 and 561-nanometer lasers simultaneously. Then, change the valve from the manual mode to injection mode to let the imaging buffer flush the protein sample into the flow cell after 60 seconds.
To remove the free EWS-FLI1, keep washing the flow cell with the imaging buffer for five minutes with only the 561-nanometer laser switched on. Then, stop the flow and incubate at 37 degrees Celsius for 10 minutes.
After 10 minutes, turn on the flow at 0.4 milliliters per minute to let the DNA extend, and acquire images at two-second intervals between different frames to obtain high-throughput data of EWS-FLI1 condensate formation.
