Abstract
Examining the membrane proteome is vital to understand their role in normal and disease function. However, the isolation and extraction of multi‐spanning membrane proteins for proteomic study often proves difficult. Traditional isolation methods are tedious and time‐consuming. Additional drawbacks include poor solubilization, incompatibility with downstream applications, and disruption of membrane protein complexes. We report a procedure that enables fast, convenient solubilization of membrane proteins from cultured cells and tissue. This sequential detergent extraction method allows for the enrichment of both integral membrane proteins and membrane‐associated proteins. To validate our method, three integral multi‐spanning membrane proteins ADP/ATP translocase 3 (SLC25A6), sodium/potassium ATPase alpha subunit (AT1A1) and adenylate cyclase 2 (ADCY2)were evaluated. Membrane protein extraction in cell lines (A431, HeLa, HCT116, HepG2, HEK293 and A549) and mouse tissue (brain, kidney and liver) using this reagent‐based extraction outperforms the other available kits as determined by western blotting and mass spectrometry. Additionally, extraction of multi‐spanning membrane protein is improved by increasing the ionic strength of the solubilization buffer, obtaining 70‐85% extraction, as confirmed by western blot densitometry. Native membrane protein complexes are also preserved, allowing for compatibility with co‐immunoprecipitation experiments. This straight forward and robust method enables researchers to better investigate the role of membrane proteins in cellular functions.
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Disclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.