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Efficient and Convenient Enrichment of Multi‐spanning Membrane Proteins for Proteomic Studies
Author(s) -
Geddes Joanna,
Meier Scott,
Cirbaite Evelina,
Siurkus Juozas,
Kaboord Barbara,
Opperman Kay
Publication year - 2015
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.29.1_supplement.567.27
Subject(s) - membrane protein , integral membrane protein , western blot , blot , proteome , immunoprecipitation , protein purification , membrane , biochemistry , chemistry , proteomics , transmembrane protein , protein subunit , microbiology and biotechnology , biology , chromatography , receptor , gene
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.