Single-Molecule Imaging to Visualize Fusion Protein Condensates Assembly on DNA Curtains

Abstract

Source: Zuo, L. et al., Single-Molecule Imaging of EWS-FLI1 Condensates Assembling on DNA. J. Vis. Exp. (2021)

This video describes a method to visualize the assembly of fusion protein condensates on DNA molecules at specific loci using single-molecule imaging. These condensates are formed due to LCD-LCD and LCD-DBD interactions, which allow for the recruitment of RNA polymerase II and enhanced gene transcription.

Protocol

1. Imaging of EWS-FLI1 condensation formation on DNA Curtains

1. Open the imaging software, find and mark the positions of the 3 × 3 zig-zag patterns under the bright field.
2. Turn on the flow at 0.2 mL/min to stain the DNA with the double-stranded DNA dye for 10 min.
3. Dilute mCherry-EWS-FLI1 with the imaging buffer to the concentration of 100 nM in 100 µL in the tube.
4. Load the protein sample through the valve with a 100 µL glass syringe, and change the flow rate to 0.4 mL/min.
5. Turn on the 488 nm laser and pre-scan each region to check the DNA distribution state; select the region where the....

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
488 nm diode-pumped solid-state laser	Coherent	OBIS488LS
561 nm diode-pumped solid-state laser	Coherent	OBIS561LS
Nikon Inverted Microscope	Nikon	Eclipse Ti
PMMA	PMMA	Prime95B