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).