title: High-Throughput Fluorometric Assay for Membrane-Protein Interaction.
abstract: Membrane-protein interaction plays key roles in a wide variety of biological processes. To facilitate rapid and sensitive measurement of membrane binding of soluble proteins, we developed a fluorescence-based quantitative assay that is universally applicable to all proteins. This fluorescence-quenching assay employs fluorescence protein (FP)-tagged proteins whose fluorescence intensity is greatly decreased when they bind vesicles containing synthetic lipid dark quenchers, such as N-dimethylaminoazobenzenesulfonylphosphatidylethanolamine (dabsyl-PE). This simple assay can be performed with either a spectrofluorometer or a plate reader and optimized for different proteins with various combinations of FPs and quenching lipids. The assay allows rapid, sensitive, and accurate determination of lipid specificity and affinity for various lipid binding domains and proteins, and also high-throughput screening of small molecules that modulate membrane binding of proteins.
authors.affiliation: Department of Chemistry, University of Illinois at Chicago, Chicago, IL, 60607, USA. wcho@uic.edu.
authors.forename: Wonhwa
authors.initials: W
authors.lastname: Cho
authors.affiliation: Department of Chemistry, University of Illinois at Chicago, Chicago, IL, 60607, USA.
authors.forename: Hyunjin
authors.initials: H
authors.lastname: Kim
authors.affiliation: Department of Chemistry, University of Illinois at Chicago, Chicago, IL, 60607, USA.
authors.forename: Yusi
authors.initials: Y
authors.lastname: Hu
mesh_terms: D002462:Cell Membrane / Q000378:metabolism*; D057166:High-Throughput Screening Assays; D008565:Membrane Proteins / Q000378:metabolism*; D011485:Protein Binding; D013050:Spectrometry, Fluorescence / Q000379:methods
chemical_list: D008565:Membrane Proteins
keywords: Dark quencher s; Fluorescence proteins; Lipid specificity, high-throughput fluorescence assay; Membrane binding inhibitors; Membrane–protein binding