AI Article Synopsis
- Protein-protein interactions (PPIs) are crucial for many biological functions, and understanding them is essential for studying how proteins work.
- The study proposes a straightforward method to assess PPI using mammalian cells, involving introduction of expression vectors, cell lysis, and protein isolation with an affinity gel.
- The researchers also suggest using affinity antibodies to confirm PPIs, highlighting the importance of maintaining protein integrity throughout the process for accurate results.
Article Abstract
Protein-protein interactions (PPIs) play a pivotal role in biological phenomena, such as cellular organization, intracellular signal transduction, and transcriptional regulation. Therefore, understanding PPIs is an important starting point for further investigation of the function of the target protein. In this study, we propose a simple method to determine the binding of two target proteins by introducing mammalian expression vectors into HEK-293 cells using the polyethylenimine method, lysing the cells in homemade protein lysis buffer, and pulling down the target proteins on an epitope tag affinity gel. In addition, the PPI between the various epitope tag fused proteins can be confirmed by using affinity antibodies against each tag instead of the epitope tag affinity gel. This protocol could also be used to verify various PPIs, including nuclear extracts, from other cell lines. Therefore, it can be used as a basic method in a variety of PPI experiments. Proteins degrade by extended time course and repeated freeze-thaw cycles. Therefore, cell lysis, immunoprecipitation, and immunoblotting should be performed as seamlessly as possible.
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| http://dx.doi.org/10.3791/66085 | DOI Listing |
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