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Updated: Dec 30, 2025
Purification of Ubiquitinated p53 Proteins from Mammalian Cells
Published on: March 21, 2022
Interaction of p53 with cellular proteins
David M Keller1, Shelya X Zeng, Hua Lu
1Department of Biochemistry and Molecular Biology, Oregon Health and Science University, Portland, USA.
Abstract:
Cellular proteins that interact with p53 play a major role in both positive and negative regulation of this tumor suppressor and can fine-tune its response to specific cellular stresses. As a consequence, p53 biology will not be complete until these interacting proteins are fully characterized. This chapter outlines two methods for identifying and characterizing p53-binding proteins: (i) glutathione-S-transferase (GST) protein-protein interaction assay; and (ii) co-immunoprecipitation (co-IP) assay. These two methods are ideal for any laboratory to perform; the assays are short in duration, do not require specialized expertise to establish in the laboratory, give reliable and reproducible data, and are cost-efficient, because few reagents are needed. This chapter gives a basic description of these two techniques and provides tips that are not found in other protocol manuals on how to achieve the best results.
Insights
Identifying p53-binding proteins is crucial for understanding its tumor suppressor functions. This chapter details glutathione-S-transferase (GST) and co-immunoprecipitation (co-IP) assays for characterizing these interactions.
Area of Science:
- Molecular Biology
- Cancer Research
- Cellular Signaling
Background:
- p53 is a critical tumor suppressor protein.
- Cellular proteins interacting with p53 modulate its regulatory functions.
- Complete understanding of p53 biology requires characterization of its interacting partners.
Purpose of the Study:
- To outline methods for identifying and characterizing p53-binding proteins.
- To provide practical guidance for implementing protein-protein interaction assays.
- To facilitate comprehensive studies of p53-mediated cellular responses.
Main Methods:
- Glutathione-S-transferase (GST) protein-protein interaction assay.
- Co-immunoprecipitation (co-IP) assay.
- Detailed protocols and optimization tips for both assays.
Main Results:
- The described methods enable reliable identification of p53-interacting proteins.
- GST and co-IP assays are shown to be cost-efficient and reproducible.
- The techniques are accessible for laboratories without specialized expertise.
Conclusions:
- Characterizing p53-interacting proteins is essential for a complete understanding of p53 tumor suppressor activity.
- GST and co-IP assays offer practical, efficient, and reliable approaches for this characterization.
- These methods can be readily adopted to advance p53 research.
