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Method Article
Identification of interacting proteins using PUP-IT
Haopeng Wang
School of Life Science and Technology, ShanghaiTech University
Corresponding Author
Min Zhuang
School of Life Science and Technology, ShanghaiTech University
Corresponding Author
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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
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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)
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This is a preprint. It has not completed peer review.
Method Article
Identification of interacting proteins using PUP-IT
Haopeng Wang
School of Life Science and Technology, ShanghaiTech University
Corresponding Author
Min Zhuang
School of Life Science and Technology, ShanghaiTech University
Corresponding Author
Badges
Prescreen
This manuscript passed our Prescreen assessment
Complete author information
Appropriate declaration statements
Potential risks to human health
Prescreen
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.
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)