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

Published: June 29, 2023

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 Open the imaging software, find and mark the positions of the 3 × 3 zig-zag patterns under the bright field. Turn on the flow at 0.2 mL/min to stain the DNA with the double-stranded DNA dye for 10 min. Dilute mCherry-EWS-FLI1 with the imaging buffer to the concentration of 100 nM in 100 µL in the tube. Load the protein sample through the valve with a 100 µL glass syringe, and change the…

Declarações

The authors have nothing to disclose.

Materials

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

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Single-Molecule Imaging to Visualize Fusion Protein Condensates Assembly on DNA Curtains. J. Vis. Exp. (Pending Publication), e21444, doi: (2023).

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