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High-Throughput Fluorometric Assay for Membrane–Protein Interaction

  • Wonhwa ChoEmail author
  • Hyunjin Kim
  • Yusi Hu
Part of the Methods in Molecular Biology book series (MIMB, volume 1376)

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.

Key words

Membrane–protein binding Lipid specificity, high-throughput fluorescence assay Membrane binding inhibitors Dark quenchers Fluorescence proteins 

Abbreviations

Dabsyl-PE

Dimethylaminoazobenzenesulfonyl-phosphatidylethanolamine

DMSO

Dimethylsulfoxide

EGFP

Enhanced green fluorescence protein

FP

Fluorescence protein

PC

Phosphatidylcholine

POPC

1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine

POPE

1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine

POPS

1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoserine

PS

Phosphatidylserine

PtdIns

Phosphatidylinositol

PtdIns(3,4,5)P3

Phosphatidylinositol-3,4,5-trisphosphate

PtdInsP

Phosphoinositides

SPR

Surface plasmon resonance

YFP

Yellow fluorescence protein

Notes

Acknowledgements

The work is supported by national Institutes of Health grants GM68849 and GM110128. We thank Prof. Daesung Lee for assisting in dark quencher lipid synthesis and Charles Delisle for preliminary work on high-throughput screening.

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Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  1. 1.Department of ChemistryUniversity of Illinois at ChicagoChicagoUSA

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