Li, Jinyuan and Zhou, Zijing and Cox, Charles (2025) Western Blotting and Immunoprecipitation of Native Human PIEZO1 Channels. BIO-PROTOCOL, 15 (1376). ISSN 2331-8325
Full text not available from this repository.Abstract
PIEZO1 is a mechanically activated ion channel essential for mechanotransduction and downstream signaling in almost all organ systems. Western blotting is commonly used to study the expression, stability, and post-translational modifications of proteins. However, as a large transmembrane protein, PIEZO1 contains extensive hydrophobic regions and undergoes post-translational modifications that increase its propensity for nonspecific protein-protein interactions. As a result, conventional sample preparation methods seem unsuitable for PIEZO1. For example, heating and sonicating transmembrane proteins exposes hydrophobic regions, leading to aggregation, improper detergent interactions, and loss of solubility, ultimately compromising their detection in western blots. To address these challenges, we developed a western blot protocol optimized for human PIEZO1 by preparing lysates consistently at lower temperatures and incorporating strong reducing and alkylation reagents into the western blot lysis buffer to ensure proper protein solubilization and minimal cross-linking. Using the same antibody, we also developed an immunoprecipitation protocol with optimized detergents to maintain the solubilization of native human PIEZO1, enabling the discovery of a new family of auxiliary subunits. Key features * Simple modifications to the standard RIPA buffer prevent protein aggregates of large transmembrane proteins. * Minimal protein degradation and cross-linking by modifying cell lysis conditions and protein extraction process. * Clear separation of glycosylated and non-glycosylated PIEZO1 by SDS-PAGE.
| Item Type: | Article |
|---|---|
| Subjects: | R Medicine > R Medicine (General) |
| Depositing User: | Repository Administrator |
| Date Deposited: | 04 Dec 2025 04:40 |
| Last Modified: | 04 Dec 2025 04:40 |
| URI: | http://eprints.victorchang.edu.au/id/eprint/1770 |
Actions (login required)
 |
View Item |