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High‐Throughput Fluorescence Assay for Membrane‐Protein Interaction
Author(s) -
Kim Hyunjin,
Afsari Hamid Samareh,
Sheng Ren,
Cho Wonhwa
Publication year - 2013
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.27.1_supplement.lb85
Subject(s) - fluorescence , green fluorescent protein , chemistry , fusion protein , biophysics , biochemistry , membrane , membrane protein , vesicle , ligand binding assay , lipid bilayer fusion , biology , receptor , gene , physics , quantum mechanics , recombinant dna
Membrane‐protein interactions play key roles in a wide variety of biological processes, including cell signaling. Although various methods have been employed to measure membrane binding of soluble proteins, a robust high‐throughput assay that is universally applicable to all proteins is lacking at present. Here we report a new fluorescence quenching assay utilizing enhanced green fluorescence protein (EGFP)‐fusion proteins and a lipid containing a dark quencher, N‐dimethylaminoazobenzenesulfonyl‐phosphatidylethanolamine (dabsyl‐PE). The EGFP fluorescence emission intensity showed a large decrease when EGFP‐fusion proteins bound the vesicles containing 5–10 mole% dabsyl‐PE. This simple assay, which can be performed using either a cuvette‐based spectrofluorometer or a fluorescence plate reader, allowed rapid, sensitive, and accurate determination of lipid specificity and affinity for various lipid‐binding domains, including two pleckstrin homology (PH) domains, an epsin N‐terminal homology (ENTH) domain, and a phox (PX) domain. The assay was also successfully applied to high‐throughput screening of small molecules that modulate membrane binding of proteins.