Single-Cell Single-Molecule Pull-Down (sc-SiMPull) for Detection of Protein Complexes from Embryonic Lysates
Naomi Stolpner1, Daniel J Dickinson2
1Department of Molecular Biosciences, University of Texas at Austin, Austin, TX, USA.
Methods in Molecular Biology (Clifton, N.J.)
|February 11, 2022
Summary
We developed single-cell, single-molecule pull-down (sc-SiMPull) to visualize individual protein complexes and their dynamics in single cells. This method reveals protein binding partners and stoichiometry lost in bulk assays.
Area of Science:
- Cellular biology
- Biochemistry
- Microscopy
Background:
- Understanding protein complex dynamics is crucial for cell signaling.
- Bulk biochemical methods obscure individual protein complex diversity.
- Single-cell analysis is needed to resolve protein complex heterogeneity.
Purpose of the Study:
- To introduce a novel method for visualizing individual protein complexes in single cells.
- To analyze protein binding partners and stoichiometry at the single-molecule level.
- To study the temporal dynamics of protein complexes during cellular signaling.
Main Methods:
- Developed single-cell, single-molecule pull-down (sc-SiMPull).
- Utilized TIRF microscopy for co-immunoprecipitation.
- Analyzed thousands of individual protein complexes from single-cell lysates.
Main Results:
- sc-SiMPull visualizes individual proteins, their binding partners, and stoichiometry.
- The method captures the diversity of protein complexes within single cells.
- Temporal dynamics of protein complexes can be constructed by iterating sc-SiMPull.
Conclusions:
- sc-SiMPull overcomes limitations of bulk methods for studying protein complexes.
- This technique provides unprecedented insight into single-cell protein complex organization and dynamics.
- sc-SiMPull enables the study of dynamic changes in protein complexes during cell signaling.
