Labelling HaloTag Fusion Proteins with HaloTag Ligand in Living Cells

@article{Duc2017LabellingHF,
  title={Labelling HaloTag Fusion Proteins with HaloTag Ligand in Living Cells},
  author={Huy Nguyen Duc and Xiaojun Ren},
  journal={Bio-protocol},
  year={2017},
  volume={7}
}
HaloTag has been widely used to label proteins in vitro and in vivo (Los et al., 2008). In this protocol, we describe labelling HaloTag-Cbx fusion proteins by HaloTag ligands for live-cell single-molecule imaging (Zhen et al., 2016). 
View on PubMed
doi.org
5 Citations
Methods Citations
1

5 Citations

CRISPR/Cas9 Genome Editing vs. Over-Expression for Fluorescent Extracellular Vesicle-Labeling: A Quantitative Analysis
TLDR
This work quantitatively compared the degree of labeling of secreted small extracellular vesicles from conventional over-expression and the CRISPR/Cas9 approach with true single-particle measurements, and fluorescently labeled EVs using the HaloTag with lipid membrane permeable dye, JaneliaFluor® 646, which allowed us to perform 3D-localization microscopy of single EVs taken up by the cultured cells.
Advances in Chromatin Imaging at Kilobase-Scale Resolution.
Live-cell single-molecule tracking reveals co-recognition of H3K27me3 and DNA targets polycomb Cbx7-PRC1 to chromatin
TLDR
A novel hierarchical cooperation mechanism by which histone modifications and DNA coordinate to target chromatin regulatory complexes is suggested by combining live-cell single-molecule tracking (SMT) and genetic engineering.
Nuclear condensates of the Polycomb protein chromobox 2 (CBX2) assemble through phase separation
TLDR
The results reveal that PcG condensates assemble through liquid–liquid phase separation (LLPS) and suggest that phase-separated condensate can organize Pcg-bound chromatin, and reveal a mechanism underlying the assembly of CBX2–PRC1 condensating.
Polycomb Cbx2 Condensates Assemble through Phase Separation
TLDR
The results reveal that PcG condensates assemble through liquid-liquid phase separation (LLPS) and suggest that P cG-bound chromatin is in part organized through phase-separated condensating.

References

SHOWING 1-8 OF 8 REFERENCES
HaloTag: a novel protein labeling technology for cell imaging and protein analysis.
TLDR
The utility of this modular protein tagging system for cellular imaging and protein immobilization is demonstrated by analyzing multiple molecular processes associated with NF-kappaB-mediated cellular physiology, including imaging of subcellular protein translocation and capture of protein--protein and protein--DNA complexes.
A general method to improve fluorophores for live-cell and single-molecule microscopy
TLDR
Inspired by molecular modeling, the N,N-dimethylamino substituents in tetramethylrhodamine are replaced with four-membered azetidine rings, which doubles the quantum efficiency and improves the photon yield of the dye in applications ranging from in vitro single-molecule measurements to super-resolution imaging.
Tracking single molecules at work in living cells.
TLDR
SMT methods are reviewed, the recent results obtained by SMT are summarized, related superresolution microscopy data is summarized, and special concerns when SMT applications are shifted from the in vitro paradigms to living cells are described.
Development of photostable fluorophores for molecular imaging.
Imaging live-cell dynamics and structure at the single-molecule level.
Dynamic chromatin technologies: from individual molecules to epigenomic regulation in cells
TLDR
Recent developments in biochemical, single-molecule biophysical and single-cell genomic technologies are discussed and how the findings from these approaches can be integrated to paint a comprehensive picture of dynamic chromatin states are reviewed.
Live-cell single-molecule tracking reveals co-recognition of H3K27me3 and DNA targets polycomb Cbx7-PRC1 to chromatin
TLDR
A novel hierarchical cooperation mechanism by which histone modifications and DNA coordinate to target chromatin regulatory complexes is suggested by combining live-cell single-molecule tracking (SMT) and genetic engineering.
Single-Molecule Fluorescence Microscopy Methods in Chromatin Biology