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Method Article
Haopeng Wang
School of Life Science and Technology, ShanghaiTech University
Corresponding Author
Min Zhuang
School of Life Science and Technology, ShanghaiTech University
Corresponding Author
License:
This work is licensed under a CC BY 4.0 License. Read Full License
Protein proximity labeling has been developed to identify protein-protein interactions. Here we report a tagging method termed PUP-IT (pupylation based interaction tagging) where a bacterial PUP ligase is fused to the bait protein and this chimeric protein mediates the covalent modification of prey protein with Pup protein. Pup is a small protein containing 64 amino acids. The N terminus of Pup can be fused to the bacteria-derived biotinylated BCCP domain. Therefore, any protein that modified by Pup can be enriched by streptavidin pull down under denaturing condition, which is often used by other types of sample preparation for mass spectrometry identification.
Here we describe two ways to achieve Pup labeling in cells. One is based on transient transfection and another based on the inducible cell line. After labeling in cells, the Pup labeled target proteins can be enriched by using the same streptavidin pull-down procedure.
Keywords
proximity labeling, PUP-IT
The authors declare no competing financial interests.
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Biochemistry
Associated Publications
A proximity-tagging system to identify membrane protein–protein interactions
by Qiang Liu, Jun Zheng, Weiping Sun, +5
Nature Methods (14 August, 2018)
An open repository of community-contributed protocols sponsored by Nature Research.
Learn more about Protocol Exchange
Method Article
Haopeng Wang
School of Life Science and Technology, ShanghaiTech University
Corresponding Author
Min Zhuang
School of Life Science and Technology, ShanghaiTech University
Corresponding Author
Version History
CURRENT STATUS: Posted
Version 1
Posted 14 Aug, 2018
No community comments so far
Metrics
Comments: 0
HTML Views: ...
Subject Areas
Biochemistry
License:
This work is licensed under a CC BY 4.0 License. Read Full License
Protein proximity labeling has been developed to identify protein-protein interactions. Here we report a tagging method termed PUP-IT (pupylation based interaction tagging) where a bacterial PUP ligase is fused to the bait protein and this chimeric protein mediates the covalent modification of prey protein with Pup protein. Pup is a small protein containing 64 amino acids. The N terminus of Pup can be fused to the bacteria-derived biotinylated BCCP domain. Therefore, any protein that modified by Pup can be enriched by streptavidin pull down under denaturing condition, which is often used by other types of sample preparation for mass spectrometry identification.
Here we describe two ways to achieve Pup labeling in cells. One is based on transient transfection and another based on the inducible cell line. After labeling in cells, the Pup labeled target proteins can be enriched by using the same streptavidin pull-down procedure.
The authors declare no competing financial interests.
Loading...
Associated Publications
A proximity-tagging system to identify membrane protein–protein interactions
by Qiang Liu, Jun Zheng, Weiping Sun, +5
Nature Methods (14 August, 2018)