Related Experiment Video
Updated: Aug 26, 2025
07:02
TurboID-Based Proximity Labeling for In Planta Identification of Protein-Protein Interaction Networks
Published on: May 17, 2020
24.5K
Off-the-shelf proximity biotinylation using ProtA-TurboID.
Irene Santos-Barriopedro1, Guido van Mierlo2, Michiel Vermeulen3
1Department of Molecular Biology, Faculty of Science, Radboud Institute for Molecular Life Sciences, Oncode Institute, Radboud University Nijmegen, Nijmegen, the Netherlands.
Nature Protocols
|October 12, 2022
Summary
We developed a new proximity biotinylation method using ProteinA-TurboID, enabling proteome identification in hard-to-transfect cells. This accessible assay identifies in vivo protein interactions within three days.
Area of Science:
- Molecular Biology
- Proteomics
- Biochemistry
Background:
- Proximity biotinylation identifies proteins near a protein of interest in vivo.
- Traditional methods require genetic manipulation (overexpression or CRISPR), limiting use in difficult-to-transfect cells.
Purpose of the Study:
- To develop an accessible, antibody-based proximity biotinylation assay.
- To enable in vivo proximal proteome identification in any cell type.
Main Methods:
- An 'off-the-shelf' recombinant enzyme (ProteinA-TurboID) was developed.
- Cells are treated with a bait-specific antibody and ProteinA-TurboID, followed by biotin addition.
- Biotinylated proteins are enriched using streptavidin beads and identified by mass spectrometry.
Main Results:
- The ProteinA-TurboID method allows for proximity biotinylation without genetic manipulation.
- The protocol is user-friendly, completable within three days.
- The method is optimized for nuclear targets but adaptable to other cellular compartments.
Conclusions:
- This antibody-based proximity biotinylation assay expands the utility of proteome identification.
- It provides a versatile tool for studying protein interactions in diverse cell types, including those resistant to transfection.
