Related Experiment Video
Updated: May 8, 2026
14:43
Combining Single-molecule Manipulation and Imaging for the Study of Protein-DNA Interactions
Published on: August 27, 2014
Single-Molecule Imaging of Endogenous Proteins.
Anusha Malapaka1, Frédéric A Meunier1,2, Rachel S Gormal3
1Clem Jones Centre for Ageing Dementia Research (CJCADR), Queensland Brain Institute, The University of Queensland, Brisbane, QLD, Australia.
Methods in Molecular Biology (Clifton, N.J.)
|May 6, 2026
Summary
This study introduces Fluorescent Intrabody Localization Microscopy (FiLM) and CRISPR/Cas9 gene-editing to track endogenous proteins in living cells. These methods enable observation of protein dynamics without artificial overexpression, providing native biological insights.
Area of Science:
- Cellular biology
- Molecular imaging
- Biophysics
Background:
- Single-molecule imaging reveals protein organization and function.
- Overexpression artifacts can distort biological observations.
- Tracking endogenous proteins in native environments is crucial.
Purpose of the Study:
- To develop and present methods for visualizing and tracking endogenous proteins in living cells.
- To overcome limitations of overexpression in single-molecule imaging studies.
- To observe protein dynamics within their native cellular context.
Main Methods:
- Fluorescent Intrabody Localization Microscopy (FiLM) utilizing nanobodies.
- CRISPR/Cas9 gene-editing for expressing tagged endogenous proteins.
- Workflow design for plasmid backbones and cell culture.
Main Results:
- Successful implementation of FiLM for single-particle tracking of endogenous proteins.
- Generation of cell lines expressing tagged endogenous proteins at physiological levels via gene-editing.
- Demonstration of a workflow for tracking native protein dynamics.
Conclusions:
- FiLM and CRISPR/Cas9 offer powerful tools for studying endogenous protein behavior.
- These methods allow for observation of protein dynamics without overexpression artifacts.
- The described workflow facilitates research into native protein organization and function.
